A conceptual model of lateral muscular tension in patients presenting thyroid associated ophthalmopathy (TAO) has been recently described. Clinical improvement has been achieved by using acupuncture on points belonging to the so-called extraordinary meridians. The aim of this study was to characterize the anatomical structures related to these acupuncture points by means of 3D MRI image rendering relying on external markers.The investigation was carried out the index case patient of the lateral tension model. A licensed medical acupuncture practitioner located the following acupuncture points: 1) Yin qiao mai meridian : Kidney 3, Kidney 6, the plantar Kidney 6 (Nan jing description); 2) Yang qiao mai meridian : Bladder 62, Bladder 59, Bladder 61, and the plantar Bladder 62 (Nan jing description); 3) Dai mai meridian (wait): Liver 13, Gall bladder 26, Gall bladder 27, Gall bladder 28, and Gall bladder 29. The points were marked by taping a nitro-glycerin capsule on the skin. Imaging was done on a Siemens Magnetom Avanto MR scanner using an array head and body coil. Mainly T1-weighted imaging sequences, as routinely used for patient exams, were used to obtain multi-slice images. The image data were rendered in 3D modus using dedicated software .Points of the Dai mai meridian – at the level of the waist – corresponded to the obliquus externus abdominis and the obliquus internus abdominis. Points of the Yin qiao mai meridian – at the medial side of the ankle – corresponded to tendinous structures of the flexor digitorum longus as well as to muscular structures of the abductor hallucis on the foot sole. Points of the Yang qiao mai meridian – at the lateral side of the ankle – corresponded to tendinous structures of the peroneus brevis, the peroneous longus, and the lateral surface of the calcaneus and close to the foot sole to the abductor digiti minimi.This non-invasive MRI investigation has revealed the anatomical relations of acupuncture points belonging to 3 of the so-called extraordinary meridians. We conclude that the clinically developed "WOMED concept of lateral tension" is related to tendino-muscular structures. Acupuncture is a fundamental component of Traditional Chinese Medicine (TCM) used in therapeutical settings. Textbooks on acupuncture describe a learning process based on the observation of changes of the surface of the body in relation to disease conditions. Based on these clinical experiences, the so-called meridians have been classically recognized and described . The namEven though acupuncture points have classical descriptions related to location, interactions and effects, the mechanisms by which acupuncture works are not fully known. Based on the pioneering work of Rasmussen and Penfield, who showed the correspondence between skin and cortical representation , centralBased on clinical observations gained from patients with thyroid associated ophthalmopathy (TAO) we have recently developed the a biomechanical model related to low-grade inflammation of the connective tissue . In thisFollowing an initial study which used gold acupuncture needles for direct MRI imaging  we have The patient is a 51 year-old male who had presented pain on the lateral sides of the right shank 1 year ago. His previous medical history revealed an episode of sudden foot inversion during eccentric muscle exercise (downhill running) which had happened 10 years ago. Two years after the traumatic event pain in the foot appeared. Both a bone scintigraphy as well as an X-ray examination was uneventful. Due to the pain, regular athletic training had been reduced in intensity in the following 5 years. Regular training for triathlon was started again three years ago; however pain reappeared on the right leg affecting the lateral aspect of the shank. On examination the skin felt warm and tense and the range of motion of the right ankle was reduced. Anti-inflammatory medication and physical medicine methods attained only temporary help. Acupuncture treatments based on meridian concepts, i.e. treatment according to the topographical distribution of acupuncture meridians, were partially successful. In spite of these therapeutic procedures, complaints re-appeared after some months. Finally the acupuncture treatment strategy was changed in order to include concepts of the so-called extraordinary meridians. Therapy consisted of needling of the Bl62 point (Shen mai) and of the Gb26 point (Dai mai) together with blood letting at the distal point of the bladder meridian, Bl67. The treatment was repeated three times after which the symptoms subsided. The skin temperature on the shank became normal and the feeling of lateral tension disappeared. The present MRI study was conducted as a control procedure. Institutional ethical approval was obtained for the study. The individual gave his consent to participate in the study.th rib). This point corresponds to the intersection jiaohui point of the foot shao yang gall bladder channel and the girdling vessel. The Shen mai point is located in the depression five fen below the outer anklebone. It corresponds to the confluence-jiaohui point of the eight extraordinary vessels (yang motility vessel)". The inclusion of the plantar level of both Kidney 6 and Urinary Bladder 62 was done based on the ancestral description of these points as can be found in the Nan jing, Chapter 28 where it can be read that these points rise from the heel. Physiologically, these areas are known to deliver important information for muscle activation involved in foot and lower limb movements [A licensed medical acupuncture practitioner (RM) localized the points in question following classical descriptions. In order to avoid magnetic interferences with the imaging equipment, external markers were taped onto each point. External fiducial markers can be used successfully for image fusion studies -19. The ovements ,22.It should be noted that the location of acupuncture points is based on a proportional measure system where the unit is the cun. One cun is defined either as "the distance between the ends of the creases of the interphalangeal joints of the middle finger at their widest point" or "the width of the interphalangeal joint of the thumb" . One tenMRI imaging was done on a Siemens Magnetom Avanto MR scanner using a head array and a body coil. 3D image sets were obtained from each region studied. Imaging of the abdominal region was done as follows: body array coil, fat-saturated T1-weighted 3D gradient-echo sequence for MRI of the body  : TR = 4.Nitro-glycerin capsules were used as external fiducial markers for the location of the acupuncture points. In previous publications we have demonstrated the utility of these capsules in MRI studies . Under TThe image data sets were rendered in 3D modus using dedicated software . Three levels of image peeling, i.e. surface to tendons and ligament levels were generated. This allowed us to look at the underlying structures. The images were analyzed by an experienced musculoskeletal radiologist specialized in MRI (AR). The anatomical structures were sought at the level that would correspond to the tip of an inserted acupuncture needle.The routine evaluation of the MRI images did not reveal any pathological changes in the subject studied. The description of the location of the acupuncture points and the anatomical structures being recognized are summarized in Table Points of the Dai mai meridian corresponded mostly to superficial muscular structures of the abdomen, i.e. the obliquus externus abdominis and the obliquus internus abdominis. Points of the Yin qiao mai meridian at the level of the ankle corresponded to tendinous structures of the flexor digitorum longus as well as muscular structures of the abductor hallucis on the foot sole. Points of the Yang qiao mai meridian at the level of the ankle corresponded to tendinous structures of the peroneous brevis, the peroneous longus, the lateral surface of the calcaneus and on the foot sole to the abductor digiti minimi.In a short historical analysis of acupuncture Fee et al.  have staThe MRI study was conducted using external fiducial markers which are otherwise needed for image fusion in different imaging modalities (e.g. MRI + PET or MRI + SPECT or MRI + CT + PET + SPECT) ,19,25-30Our recent description of the WOMED concept of lateral tension as a mechanism related to low level inflammation of the connective tissue in patients with thyroid associated ophthalmopathy (TAO) included the diagnostic-therapeutic use of specific acupuncture points that belong to the so-called extraordinary meridians . Based oFigure th costal cartilage, halfway between the iliac crest and the rib cage..." [Besides the anatomical correlates discussed above, we would like to mention some relevant biomechanical data. Anticipation of limb movement involves the activation of the trunk and abdominal muscles . This accage..." . Furthercage..." , thus sucage..." . Alteredcage..." . In addicage..." . We havecage..." . Evidenccage..." . This phcage..." ,47-53. Icage..." ,54.Surface rendering procedures of MRI data sets together with external fiducial markers can be used successfully to describe the in-vivo anatomical relations of acupuncture points. Our data suggest a close relation of acupuncture points of the Yang and Yin motility vessels as well as of the Dai mai to tendino-muscular structures. Biomechanical data point out their importance in posture and locomotion. New examination and therapy procedures based on the "WOMED concept of lateral tension" might be of benefit in clinical practice.The author(s) declare that they have no competing interests.All authors contributed equally to this work: HM and RM have developed the concept of lateral tension, RM did the acupuncture work. AR, an experienced musculoskeletal radiologist, did the image interpretation. CK an experienced radiology physicist made and processed the MRI studies.The pre-publication history for this paper can be accessed here:


Alexandrium tamarense to elucidate dinoflagellate evolution. Previous analyses of these data have clarified plastid origin and here we study the gene content, annotate the ESTs, and analyze the genes that are putatively involved in DNA packaging.Dinoflagellates are important marine primary producers and grazers and cause toxic "red tides". These taxa are characterized by many unique features such as immense genomes, the absence of nucleosomes, and photosynthetic organelles (plastids) that have been gained and lost multiple times. We generated EST sequences from non-normalized and normalized cDNA libraries from a culture of the toxic species Approximately 20% of the 6,723 unique  ESTs data could be annotated using Blast searches against GenBank. Several putative dinoflagellate-specific mRNAs were identified, including one novel plastid protein. Dinoflagellate genes, similar to other eukaryotes, have a high GC-content that is reflected in the amino acid codon usage. Highly represented transcripts include histone-like (HLP) and luciferin binding proteins and several genes occur in families that encode nearly identical proteins. We also identified rare transcripts encoding a predicted protein highly similar to histone H2A.X. We speculate this histone may be retained for its role in DNA double-strand break repair.This is the most extensive collection to date of ESTs from a toxic dinoflagellate. These data will be instrumental to future research to understand the unique and complex cell biology of these organisms and for potentially identifying the genes involved in toxin production. Symbodinium, are central contributors to the health of reef ecosystems as the symbionts of corals . The consensus secondary structures were used in the comparison to the know structure of E. coli HU (PDB ID: 1MUL).The Approximately Unbiased test was done using CONSEL . ML treeJDH constructed the cDNA libraries and did the sequence and phylogenetic analyses, the Blast and GO analyses on the EST dataset, the histone and HLP analyses, and drafted the manuscript. TES did many of the other global sequence analyses of the EST dataset. HSY contributed intellectually to the manuscript. Library construction and high-throughput EST sequencing was done in the laboratory of MBS and was supervised by MFB. The bioinformatics group led by TLC did the EST sequence processing and clustering. DB conceived of and supervised this study and contributed to the manuscript. All authors read and approved the final manuscript.


Zip1 zinc transporter as a tumor-suppressor gene is described.The genetic and molecular mechanisms responsible for and associated specifically with the development and progression of malignant prostate cells are largely unidentified. In addition, despite its implication in virtually all malignant cells, the role of altered cellular metabolism as an essential factor in prostate malignancy has been largely ignored. Moreover, the intermediary metabolism of normal prostate as well as malignant prostate cells is among the least studied and most poorly understood of all mammalian cells. Some important factors, especially the role of zinc, have been identified and implicated in the development and progression of prostrate malignancy. In this review, we provide a current and updated integrated assessment of the relationships of intermediary metabolism in normal prostate and in prostate cancer. The experimental and clinical evidence that leads to the formulation of concepts of normal and malignant prostate metabolism is presented. The evidence for a concept of zinc as a tumor suppressor agent and The specialized function of the normal prostate glandular epithelium to produce and secrete enormously high levels of citrate involves and requires unique intermediary metabolism activities that are not generally associated with other normal mammalian cells. The accumulation of zinc by these cells is an essential factor in this unique metabolic relationship. In malignancy, the normal zinc-accumulating citrate-producing epithelial cells are metabolically transformed to citrate-oxidizing cells that lose the ability to accumulate zinc. A genetic alteration in the expression of ZIP1 zinc transporter is associated with this metabolic transformation. These genetic/metabolic relationships have important consequences on citrate-related metabolism, bioenergetics, cell proliferation and invasive capabilities of the malignant cells, which result in tumor-suppression characteristics.The genetic/metabolic relationships in normal prostate glandular epithelium are driven by the unique function to accumulate and secrete citrate. The genetic/metabolic transformation of the prostate malignant cells is driven by the metabolic/bioenergetic, growth/proliferative, and invasive/migration requirements of the malignant process. Zinc is critical to these relationships. An understanding of these genetic/metabolic relationships provides new directions and opportunities for development of regimens for the prevention and treatment of prostate cancer. Important insight into the genetic/metabolic requirements of the prostate malignant process is now evolving. Most importantly at this time, an appreciation and recognition of the genetic/metabolic significance and implications in the development of prostate malignancy is imperative; and much needed research in this area is essential. Hopefully, this review will help to achieve these goals. About 230,000 males are expected to be identified with prostate cancer in the next year that will result in about 30,000 deaths. Despite the extensive clinical and experimental studies over the recent decades, the pathogenesis of prostate cancer remains unknown. The genetic and molecular mechanisms responsible for and associated specifically with the development and progression of malignant prostate cells are largely unidentified ,2 for re. The comZIP1 zinc transporter as a tumor-suppressor gene will be described. The clinical/translational application of these relationships is also presented.This review will provide a current and updated integrated assessment of the relationships of intermediary metabolism in normal prostate and in prostate cancer. Much of the description of important contributions of earlier investigators and early studies that should be recognized is presented in our previous reviews -12; and The totality of this presentation is intended first to be informative. Hopefully, the presentation will be stimulating and even provocative; so that more attention is directed to this subject matter and other investigators might become involved. Finally, we hope to convince the clinical and scientific community-at-large of the importance of metabolic relationships in the development and progression of prostate cancer, and the relevance to new approaches to detection, treatment, and perhaps prevention of prostate cancer.The following are important generalizations that we consider to be axiomatic.1). In all cells, the existing cellular intermediary metabolism provides the bioenergetic/synthetic/catabolic requirements that are essential for the manifestation of the cells' current activities .2). When a cell's activity changes, its metabolism must also be adjusted consistent with any newly established bioenergetic/synthetic/catabolic requirements3). Malignant cells exhibit a parasitic existence. They have no specialized function other than the activities essential for their generational propagation (growth and proliferation), which occurs at the expense of their host.4). Malignant cells are derived from normal cells that have undergone a genetic transformation to a neoplastic cell phenotype that is endowed with malignant potential.5). Manifestation of the malignant potential of the neoplastic cell necessitates alterations in its metabolism (ie a metabolic transformation) to provide the bioenergetic/synthetic requirements of malignancy.6). In the absence of the metabolic transformation, the neoplastic cell will not progress to complete malignancy. Conversely, the metabolic transformation, in the absence of the genetic transformation to a neoplastic malignant cell, will not cause malignancy.7). Common to all malignant cells is the metabolic requirement for de novo lipogenesis/cholesterogenesis for membraneogenesis that is essential for their proliferative existence.These axioms define a relationship represented in figure The human prostate gland is a complex organ comprised of different zones that have different embryological origin and different functional activities. The focus of this presentation is the peripheral zone, which comprises about 70% of the gland, and is the major functional component. Most importantly, this is the major region of malignancy. The primary function of the prostate gland is the production and secretion of prostatic fluid -12. The The function of prostate citrate production is achieved by the activity of highly specialized glandular epithelial cells that have evolved for the capability to accumulate and secrete citrate, which we refer to as "net citrate production". This capability does not exist in other mammalian cells. Consequently the intermediary metabolism of these "citrate-producing" cells is "driven and modified" by their specialized function of net citrate production figure . In typiTo meet the precursor requirements for continued citrate synthesis, the prostate epithelial cells possess unique glucose and aspartate relationships. These cells, unlike most other mammalian cells, utilize glucose via a high aerobic glycolysis -17 that For the production of OAA, aspartate is accumulated in the prostate cells and is transaminated via mAAT and coupled to GDH activity in a pathway that we define as the aspartate-glutamate-citrate pathway figure . In thesThe relationships described above involve the metabolic adaptations that are essential to facilitate citrate synthesis. The accumulation of citrate is dependent upon its rate of synthesis versus its rate of utilization. The prevention of citrate oxidation by the prostate cells is the key relationship that is responsible for net citrate production. The prostate peripheral zone accumulates extremely high levels of zinc in the range of ~3000–5000 nmols/gram as compared to other tissues being ~200–400 nmols/gram. The cellular accumulation of zinc results in high levels of mitochondrial zinc that inhibit m-aconitase activity and citrate oxidation . That ziNet citrate production has significant bioenergetic implications figure . CompareIt is now well established that citrate and zinc levels are markedly decreased in malignant versus normal prostate tissue. These biochemical changes occur early in the development of malignancy. Moreover, malignant prostate tissue virtually never retains the characteristic high levels of citrate and zinc found in normal peripheral zone. In the absence of high cellular zinc levels, m-aconitase activity is no longer inhibited and citrate oxidation proceeds via the Krebs cycle figure . Thus weAn important benefit of this metabolic transformation is the bioenergetic relationship in that citrate oxidation provides the additional 24ATP from complete glucose oxidation that were lost by incomplete glucose oxidation in net citrate production. Thus, the malignant cells are bioenergetically more efficient than the specialized normal prostate epithelial cells. This is in contrast to the general tumor metabolism relationship in which the tumor cells are metabolically transformed to high aerobic glycolysis -7. FigurIn mammalian cells, citrate is synthesized in the mitochondria where it is either oxidized via the Krebs cycle or it is exported to the cytosol figure . In the This requires another metabolic adaptation for the specialized function of citrate secretion. As shown in figure As already described, the malignant prostate cells undergo a metabolic transformation to citrate oxidation. However, for the malignant cells to proliferate they must also adapt their metabolism for the utilization of citrate for de novo lipid biosynthesis. The initiating steps in this pathway are the export of citrate into cytosol followed by its conversion to acetyl coA. Therefore, an essential metabolic adaptation in the malignant prostate cells must be an increase in ATP citrate lyase activity. Halliday et al  observedThe available evidence indicates that, in prostate malignant cells, the mitochondrial citrate pool is utilized for oxidation via the Krebs cycle and for export to cytosol for utilization in de novo lipid biosynthesis. This is in contrast to the prevailing concept of tumor cell metabolism of a "truncated Krebs cycle" as proposed by Parlo and Coleman ,27. The The above description presumes that citrate is the essential source of cytosolic acetyl coA for de novo lipogenesis/cholesterogenesis in malignant prostate cells. However, the possibility exists that acetate derived from circulation might be an alternative precursor for acetyl coA production. This would require the existence of cytosolic acetyl coenzyme A synthetase (AceCS1), which is up-regulated in some lipogenic mammalian cells -35. It iThe following presents a significant translational clinical application of the citrate metabolic relationship. A major current problem involved in prostate cancer (PCa) is the absence of sensitive, accurate, and preferably non-invasive procedures for the diagnosis of PCa. Moreover procedures are needed that will permit the early detection, staging, location, and estimation of the volume of the malignancy; and preferably a mapping of the prostate for follow-up of progression and regression of the malignancy. The unique citrate metabolism relationships of the prostate, coupled with recent developments and technological advancements in 1-H MRSI for the in situ determination of citrate levels, provides an excellent diagnostic/detection procedure which can achieve all these goals. In addition, the mapping of malignant loci permits accurate and precise MRSI guided radiotherapy and chemical seeding in targeting and concentrating the treatment at the malignant tissue. There now exist strong, compelling basic and clinical studies in support of the employment of MRS measurements of citrate and other associated metabolites in the diagnosis of PCa and in the in situ visualization of malignant loci -40; for ; for 40;The combination of magnetic resonance imaging (MRI) and MRSI results in a metabolic-anatomic visualization (ie a metabolic map) of the prostate gland. Figure Figure Any suggestion of tumor suppression in prostate cancer by an agent and/or a gene must have a strong and rational clinical as well as experimental basis. The following enumerates the supporting information regarding zinc suppression of prostate malignancy. As already emphasized, malignant prostate tissue is characterized by a dramatic decrease in zinc level as compared to extremely high zinc levels in normal and hyperplastic glands. Most importantly, malignant tissue rarely, if ever, exhibits the high zinc content that characterizes nonmalignant prostate glands. The consistency of these relationships is further represented in Table The inability of malignant cells to accumulate zinc is not, in itself, evidence of a tumor-suppressive effect of zinc. There must be associated effects and consequences of zinc that are inhibitory to the malignant cells. Such anti-tumor consequences do exist and can be characterized as follows.a. Metabolic/bioenergetic effects. As already described, zinc accumulation in prostate cells truncates the Krebs cycle and inhibits citrate oxidation. In addition, high zinc levels also inhibit the respiration and terminal oxidation of prostate mitochondria . These mb. Growth/proliferation effects. The accumulation of zinc inhibits growth and proliferation of prostate cells . We repoc. Invasive/migration effects. Zinc has also been shown to inhibit the invasive capabilities of malignant prostate cells. Ishii et al.  reportedThese effects of zinc accumulation are inhibitory to and incompatible with the prostate malignant process and can be defined as "tumor suppressor" effects of zinc. The depletion of zinc that occurs in malignant prostate cells in situ eliminates these anti-tumor effects. Under these conditions the neoplastic cell is able to meet the metabolic/bioenergetic requirements of malignancy, is able to grow and proliferate, and is able to invade the host tissue and metastasize.The critical issue becomes the mechanism involved in the unique ability of normal prostate cells to accumulate high zinc levels; and the mechanism involved in the lost ability of malignant cells to accumulate zinc. For an extensive review of zinc and zinc transporters in prostate cells see . The intZIP1 gene expression, the decrease in ZIP1 transporter protein, and the depletion of cellular zinc levels in malignant versus nonmalignant glands. Thus, the clinical and experimental evidence are corroborative of the role of ZIP1 and zinc changes associated with the transformation of normal glandular epithelial cells to malignant cells. Consequently, as long as ZIP1 transport activity exists and results in high zinc accumulation, the tumor suppressor effects of zinc will prevent the malignant activities of the neoplastic cells. ZIP1 (SLC39A1) can be described as a tumor suppressor gene in prostate cancer.Therefore, we propose that However, all of the above studies were conducted in prostate culture cells under in vitro conditions. An essential missing link was the relationship in normal and malignant prostate glands in situ. The first clue was derived from the report of Rishii et al  who founZIP1 expression in the neoplastic/malignant cell in situ. In contrast to its down-regulation in the malignant prostate glands in situ, Zip1 is expressed in human prostate malignant and nonmalignant cell lines thus far examined [ZIP1 is a tumor suppressor gene in prostate cancer is similar to the reported involvement of SLC5A8 as a tumor suppressor gene in colon and other cancers [SLC5A8 encodes a monocarboxylic acid transporter protein, and its down regulation results from epigenetic silencing. The specific role of the transporter in tumor suppression is not established. Ganapathy et al [This leads to the issue of the mechanism of down regulation of examined ,51,56-58 cancers -65. SLC5hy et al  propose The above discussion leads to a rational expectation that the restoration of zinc accumulation in the malignant prostate cells should arrest the malignancy and cause the death of the tumor cells. Moreover, if zinc accumulation is restored in the neoplastic/premalignant stage figure , overt mAt this point, we cannot ignore the one dot that has not yet been connected; which is the composite results of epidemiologic studies. Several studies that attempt to relate the use of dietary zinc supplementation to prostate cancer have been reported. The results and conclusions of these studies have varied from zinc supplement use having no effect; zinc supplement use being preventative against prostate cancer; and zinc supplement having harmful exacerbating effects on prostate cancer. These studies and the complex and multivariate factors that impact them are the subject of our recent review ; and wilThis provides a basis for consideration of regimens that can achieve the uptake and accumulation of zinc in neoplastic/malignant cells and can provide tumor-suppressive effects against prostate cancer.a. Genetic manipulation to restore the expression of ZIP1. Presently this is not a practical approach.b. Genetic manipulation to "activate" expression of alternative zinc uptake transporters. Presently this is not a practical approach.c. Eliminate or modify the environment conditions of the prostate gland that initiate the epigenetic silencing of Zip1 gene expression. This is dependent upon the elucidation of the mechanism responsible for the down regulation of ZIP1. Once this issue is resolved, this could be a plausible approach.d. Increase the bioavailability of zinc. This has been the basis for the common use of dietary supplemental zinc as described above. In the elderly male population, supplemental zinc would be efficacious in maintaining a normal plasma zinc level. This should be incorporated into or along with any other interventions. It is also possible that an elevated plasma zinc level could result in zinc uptake by the malignant cells in the absence of ZIP1. This would be dependent upon an elevation of plasma low molecular weight zinc-ligands that become available for cellular uptake ,11.e. Incorporation of agents that will deliver and transport zinc into the malignant cells. This is based on the use of ionophores such as sodium pyrithione to deliver cations into cells. The lack of cation specificity and cell specificity might impose toxic effects, which would need to be addressed.f. Use of m-aconitase inhibitor. A major effect of zinc is its inhibition of m-aconitase activity and citrate oxidation. In its absence, an alternative m-aconitase inhibitor could be employed. Fluoroacetate (→ fluorocitrate) is such an inhibitor. However it is not cell specific and is toxic to other mammalian cells. The development of a prostate cell-specific agent or a cell-specific chaperone for delivery could provide the inhibitory effect on citrate oxidation in the malignant cells.These are some approaches that are based on the zinc relationships. Additional approaches exist that are based on other aspects of prostate metabolism. For example, inhibition of lipogenic reactions such as ATP citrate lyase  and AceCThe clinical and experimental evidence leads to the following genetic/metabolic relationships in prostate cancer.1. The unique function of high citrate production and secretion by human prostate glands dictates the metabolic activity and associated genetic requirements of the highly specialized glandular epithelial cells. The unique accumulation of zinc by these cells is a requirement for the achievement of this function.2. The bioenergetic/synthetic, growth/proliferation, and invasive/migration requirement of malignancy dictates the metabolic activity and associated genetic requirements of the malignant cells.3. The malignant process requires the genetic and metabolic transformation of normal zinc-accumulating citrate-producing normal cells to citrate-oxidizing malignant cells that lose the ability to accumulate zinc.4. The accumulation of zinc exhibits tumor-suppressor effects that are incompatible with the process and activities of prostate malignancy.ZIP1is a tumor-suppressor gene in prostate cancer.5. The expression of ZIP1 zinc uptake transporter is responsible for zinc accumulation in prostate cells. ZIP1 is down regulated in the malignant cells in situ, which causes the depletion of zinc and elimination of its tumor-suppressor effects. Therefore we propose that 6. The restoration of high zinc levels in premalignant/malignant prostate cells will arrest and/or abort prostate malignancy.7. These relationships can serve as the basis for new approaches to the treatment and prevention of prostate cancer.8. Genetic/metabolic relationships in prostate malignancy are beginning to provide an understanding of the onset and progression of prostate cancer. Much more research in this critical area is required.RBF and LCC conceived the review, contributed to the writing of the manuscript; and read and approved the final manuscript


The cytotoxicity of two antineoplastic drugs, cisplatin and CCNU, was evaluated in Chinese hamster V79 multicell spheroids using the drugs as single agents or combinations. Cells obtained from different depths within spheroids 550-750 microns in diameter showed different sensitivities to the two agents; the external cells of the spheroids were more sensitive than the internal cells to cisplatin, whereas the internal cells were most effectively killed by CCNU. Combining the two agents produced the expected 'complementary' activity, and in addition, synergism was observed between the drugs at exposure levels practical for clinical use. For the combination treatments, both the net pattern of cell killing through the spheroid and the degree of interaction between the agents (quantified using the combination index method) were a function of the dose ratio of the two drugs, and of overall treatment intensity. BCNU produced patterns of cell killing similar to CCNU, but showed little interaction with cisplatin. Our results suggest significant clinical potential in using CCNU with cisplatin, particularly since CCNU-cisplatin combinations were synergistic even in the cell subpopulations most resistant to each drug as a single agent.


We have recently succeeded in producing transgenic mice carrying a hybrid gene consisting of mouse metallothionein promoter-enhancer and the ret oncogene (MT/ret). . A retroperitoneal tumour developed in one of 17 MT/ret transgenic founder mice. Histological analysis revealed that the tumour consisted of undifferentiated neuroblasts and differentiated ganglion cells, the latter of which were strongly positive for neuron specific enolase. Expression of the ret transgene was observed at high levels in RNA from the tumour, but not in those of other normal tissues. In addition, a 100kDa ret protein was detected in the cell lysate of the tumour. Taken together with our previous data, these results suggest a possible role for the ret oncogene in the proliferation of neural crest cells.


Health- and illness-related cognitions of pediatric patients with asthma or somatization and of their caregivers are considered relevant for patient education and for cognitive-behavioral interventions. This study investigates the relationship between diagnosis and illness perception by child and parent in two different chronic conditions such as somatization disorder and asthma.25 patients with somatoform disorders and 25 patients with asthma bronchiale completed the Giessen Complaint List and the Multidimensional Health Locus of Control Scale. Primary caregivers independently answered parallel proxy-report instruments. Analyses of variance were performed to determine the impact of diagnosis and perspective. Correlations were calculated to determine the concordance between patient and caregiver reports.No statistically significant differences in illness locus of control beliefs were found between asthma and somatoform disorder children or parents. Parents reported more internal and fatalistic locus of control beliefs compared with their children. Correlations between patient and caregiver reports of symptoms and health locus of control beliefs were low to moderate.Clinicians should take into account a sense of insufficient symptom control in both diagnostic groups and different viewpoints of patients and their parents. Somatoform disorders and asthma bronchiale are two of the most frequent chronic conditions in childhood and adolescence with prevalence rates between 2.7 per cent  and 13.1The patients' perception and evaluation of somatic symptoms are considered important for subsequent illness behavior and coping with the disease. Somatoform disorders are characterized by a specific way of exaggerated attention to and negative evaluation of bodily sensations ,9. WhereInternal locus of control represents the belief that one's own behavior is regarded as important for one's state of health. This attitude is regarded as an essential pre-condition for active coping strategies both in patients with somatoform disorder and asthma. Social externality means that powerful others, for example parents or physicians, are considered as important for symptom control. This concept triggers the help-seeking behavior of patients. Patients with fatalistic health locus of control beliefs are convinced that their health state is influenced by fate, luck or random events. Fatalistic expectations are considered maladaptive in both somatoform disorder and asthma because of the associated passive patient behaviour [The locus of control construct refers to the subjective beliefs of control that patients have over illness and health. Applying Rotter's social learning theory  to patieehaviour .In pediatric patients, the responsibility of parents for the illness behavior of their children and the caregivers' impact on the development of the children's own subjective health concepts have to be considered. Parents monitor their children's health state, decide whether medical care is to be sought and comply with medical recommendations or not. The children's and adolescent's illness-related perceptions may be influenced by parental models and suggestions ,16. On tTo our knowledge, so far no studies have compared the health locus of control beliefs of children and adolescents with somatoform disorder and with asthma bronchiale, and the corresponding parental perceptions and cognitions. The comparison of subjective illness representations would contribute to answer the question whether disease-specific cognitions have to be addressed in cognitive behavioral interventions. Moreover, the comparison of patients' and their caregivers' perceptions would allow differential planning of family-oriented psychoeducational interventions. In this exploratory study, two research questions are addressed:1) Do patients and their parents develop disease-specific health- and illness-related locus of control beliefs? As medical treatment in combination with the patients' active role in symptom control is effective for asthma but not for somatoform disorder, we would expect more social external and internal health locus of control beliefs in the asthma patients and their caregivers, compared with patients with somatoform disorder and their caregivers.2) Are parental proxy-reports and patients' self-reports on somatic complaints and locus of control beliefs correlated? In accordance with previous findings in pediatric recurrent abdominal pain  and seveInformed consent of caregivers and assent of children and adolescents to participate in the study were obtained in accordance with the principles of the local ethical committee. The assessment was done by a researcher independently from the physicians and therapists responsible for the patients' treatment. All participants filled in standardized questionnaires, the caregivers provided additional information about individual illness history, and socio-demographic data. Patients and caregivers filled in the questionnaires independently from one another.p < .05 in socio-demographic variables such as age, gender, family constellation, socio-economic status or school absence. All patients were Caucasians.Patients between 8 and 18 years of age with a clinical diagnosis of somatoform disorder or asthma bronchiale and their primary caregivers were included . This meviously . In the Multidimensional Health Locus of Control Scale  the degree of assent to each statement has to be indicated by the respondent. The questionnaire has been developed and validated with different clinical and healthy groups of children, adolescents and adults. It has good reliability and validity. In our study, the internal consistency was α = .68 for the socio-external scale, .85 for the fatalistic-external scale, and .87 for the internal dimension.The ion: KKG ) measureKKG for the perspective of caregivers by rewording the items, for example "If my son/daughter has complaints, we ask somebody for advice" instead of "If I have complaints, I ask somebody for advice." Cronbach's α for the parent form was .66 for the socio-external scale, .72 for the fatalistic external scale and .79 for the internal locus of control dimension.In order to receive additional parental reports, we modified the Pearson correlations between self-reports and parental proxy-reports in corresponding scales of the questionnaires were calculated to determine inter-rater concordance. We also calculated intra-class correlations, and the resulting scores were approximately the same as indicated by the Pearson correlations. To avoid redundant information, only Pearson coefficients are reported. A significance level of α = .05 was chosen. With regard to the explorative character of the study and to reduce the risk of β-errors, no adjustment of significance level for multiple testing was made.A descriptive analysis of the different scores was performed separately for both clinical groups and both informants. There were no statistically significant differences in socio-demographic and clinical characteristics between the two diagnostic groups. A series of 2 × 2 ANOVARs was computed with diagnosis  as between-subject factor and different dimensions of self-reported and caregiver-reported locus of control beliefs as repeated measures within-subject variables. Giessen Complaint List for Children and Adolescents . On thefatalistic locus of control perception in the asthma group , whereas the least manifestation of health locus of control was found in the asthma patients internal dimension . No significant differences in health locus of control estimations were demonstrated between the diagnostic groups , compared to less frequent internal  and even lesser socio-external health control beliefs . In contrast, there were similar levels of each control attribution in patients .The most prominent manifestation of health locus of control perceptions was the parental total score and in the cold subscale, demonstrating a parental under-estimation of children's subjective symptoms in the somatization group and a parental over-estimation of symptoms in the asthma group.Correlational analyses demonstrated weak associations between patients' and parents' reports, both separately within the clinical groups and in the total study sample  on subjective illness-related cognitions and compares patients' and their caregivers' health- and illness-related symptom perceptions and locus of control evaluations.The analysis of the locus of control beliefs demonstrate differences between parental and patients' cognitions, but not between patients or parents of different diagnostic groups. Across both diagnostic groups parents have more pronounced control beliefs than their children. This finding is significant for the internal and fatalistic health locus of control dimension, whereas only a statistical trend occurs in the difference between caregivers and patients in the socio-external control attribution.The attribution of symptoms to fate or chance indicates a sense of lack of control on illness, and consequently high scores of fatalistic-external locus of control are rather expected correlated with passive illness behavior and non-adherence to treatment. According to our results, especially parents of children and adolescents with asthma develop high fatalistic beliefs, indicating a lack of subjective predictability of their children's asthma symptoms so far. This might be due to the long history of ineffective asthma treatment in our study sample. However, the asthma patients themselves report no significantly elevated fatalistic-external control perceptions compared with their internal or socio-external attributions of symptom control. In another study with the same instrument Schmitt et al.  found evAnother relevant finding is that the asthma patients in our study sample reported low scores of perceived internal controllability of symptoms. Internal health locus of control has been demonstrated as associated with adherence to treatment  and shouThe differences between the patients' and their caregivers' health related locus of control beliefs might also be explained by developmental differences of cognitive functioning. The belief that health cannot be controlled and that fate or chance might be responsible for staying healthy or becoming ill, requires the awareness of limited personal power. Due to their tendency towards concrete and rather egocentric thinking, children may overestimate their personal impact or the influence of powerful others on their health.Our results demonstrate the ubiquity of subjective illness concepts across both chronic conditions. These findings are consistent with those of Perrin and Shapiro  who founWith regard to our second research question, we demonstrated that accordance between parents and patients is limited both in terms of symptom reports and cognitive illness concepts. Symptom reports were correlated only moderately. Consistent with the different diagnoses, children and adolescents with somatization disorder reported significantly more symptoms than peers suffering from asthma bronchiale. However, this judgment is not supported by the parents, who reported a similar level of symptoms in both clinical groups. Several explanations for this discrepancy between self reports and parent reports are possible. First, the ability of caregivers to recognize and report internal perceptions of their children reliably may be limited . PatientIn summary, the results of our study demonstrate substantial differences between patients' and parental illness-related perceptions, whereas subjective illness concepts varied independently of diagnosis.Several limitations of this study should be mentioned. First, the results have to be considered as preliminary because of its possible selection bias. The participants with psychosomatic disorder may have been altered in their health- and illness-related cognitions by impact of psychological or psychiatric diagnosis and/or treatment compared with non-referred psychosomatic patients. The participants with asthma were referred to a specialized inpatient rehabilitation program, therefore they may have been more resistant against basic patient education and counselling than average pediatric asthma patients in outpatient settings. It can be assumed that our asthma sample represents rather non-responders to treatment within primary care, so these patients might be more difficult to treat and even more altered in their health perceptions and illness beliefs. With regard to these limitations due to selection, our results cannot be generalized to all patients with somatoform disorder or asthma.Secondly, the small sample size may have concealed small between-group differences because of a limited statistical power. Large scale studies comparing different diagnostic subgroups might be able to detect differences that did not occur in our study because of its restricted sample size.Moreover, the findings in this study are based on standard self-report measures. The socio-external health locus of control scale suffered from sub-optimal internal consistency of α < .70 and should therefore be interpreted cautiously. Future studies should integrate semi-structured interviews and a qualitative methodology to collect more detailed information on the structure of subjective illness perceptions. Also possible mediating factors on symptom perception and illness concepts such as anxiety should be integrated in future study designs.However, our preliminary results contribute some interesting findings to the emerging literature on symptom perception and cognitive aspects of chronic pediatric conditions. For the first time, we evaluated the health locus of control perceptions of children with Somatizationand their parents, and found similar cognitions as found in pediatric asthma. Future studies should include larger samples in different clinical settings and multi-method designs. Longitudinal studies would be necessary to get information on changes of subjective illness perceptions and cognitions.Some clinical implications emerge from our findings. Our results indicate the relevance of multi-informant strategies in the diagnosis of health- and illness-related perceptions and cognitions in pediatric patients. Collecting parallel self-reports and caregiver reports of symptoms and health locus of control attributions should be an obligatory part of diagnosis. Such a comprehensive diagnosis is useful for planning patient education and counselling. Our findings highlight the importance of exploring patients and their caregivers separately to get valid information on subjective symptoms and illness concepts. Patients themselves may be regarded as more reliable informants about somatic symptoms, whereas both the patients' and their caregivers' subjective illness concepts are relevant for targeting cognitive-behavioral interventions. The degree of patient-parent accordance is an additional relevant aspect of diagnosis and intervention planning. Clinicians have to consider different subjective viewpoints of chronically ill children and their caregivers, and cognitive-behavioral interventions have to take into account these different health and illness concepts.The author(s) declare that they have no competing interests.LG conceived in the study, participated in its design and coordination, performed the statistical analyses and drafted the manuscript. SB participated in the design, collected the data, and helped to analyse the data. Both authors read and approved the final version of the manuscript.


Ifosfamide is clinically used in combination chemotherapy regimens for the treatment of patients with high-grade lymphomas, sarcomas and metastatic germ cell tumours. In order to reduce the oxazophosphorine-related urothelial toxicity, sodium mercaptoethane sulphonate (mesna) is used in different schedules following the administration of ifosfamide. The proposed mechanism of mesna activity is the binding of toxic oxazaphosphorine metabolites such as acrolein in the urine of the patients. Since an influence of mesna on ifosfamide anti-tumour activity is controversial, the current study has used xenografts from two human testicular cancer cell lines heterotransplanted into nude mice to study the anti-tumour activity of ifosfamide in combination with different dosages and schedules of mesna. In both human testicular cancer cell lines, H 12.1 and 2102 EP, ifosfamide demonstrated anti-tumour activity as a single agent. No reduction in ifosfamide activity was observed with the application of mesna at a dose range from 50% to 200% of the ifosfamide dose. Furthermore, the application of mesna before and 3 h after ifosfamide, a schedule used in many clinical protocols because of the short half life of mesna, not only maintained high ifosfamide anti-tumour activity but also seemed to be associated with the lower systemic and urothelial toxicity of ifosfamide therapy compared with ifosfamide given alone. In conclusion, the experimental in vivo system using human heterotransplanted testicular cancer cell lines confirms the significant anti-tumour activity of ifosfamide in malignant germ cell tumours and demonstrates that mesna does not impair ifosfamide anti-tumour activity in this model. These results are most likely transferable to the use of mesna in patients with metastatic testicular cancer.


Venous gangrene (VG) is defined as a clinical triad of skin necrosis and discolouration, documented evidence of venous thromboembolism (VTE) and presence of palpable or doppler- identifiable arterial pulsation. Venous gangrene is rare condition which is associated with poor prognosis in cancer patients. The pathogenesis of VG is multifactorial and could paradoxically be due to warfarin treatment. Heparin Induced Thrombocytopenia (HIT) associated venous gangrene develops when heparin therapy is discontinued and warfarin therapy initiated or continued.It has been reported that the presence of anticardiolipin antibodies appears to double the risk of thrombo-embolic events in cancer patients in comparison with those who are anticardiolipin antibody negative. The presence of anticardiolipin antibodies is therefore a warning sign for venous gangrene in cancer patients. Hypercoagulable state associated with malignancy, cancer treatment, prolonged immobilisation, surgical operations and metabolic syndrome are all associated with increased risk of VTE and VG.The current evidence suggests that cancer patients are at increased risk from recurrent venous thrombosis and venous gangrene, and LMWH provides potential promise as a safe and effective measure in the management of such patients. Venous gangrene (VG) is a rare condition in association with malignancy but carries a grave prognosis . Venous Interestingly, VG could paradoxically be due to warfarin treatment in association with decreased level of protein C . The epiCurrently the incidence of VG in association with cancer is not well established. However, there are a few reported cases in the literature showing that VG is always in association with venous thrombo-embolism (VTE). The annual incidence of VTE in a cancer population is 500 in 100,000 (one in two-hundred) in comparison with 117 in 100,000 in the general population . Rates oIn their study of 1041 patients with solid tumours admitted to 3 major medical centres in the USA, Sallah et al found the highest rates of VTE in cases of advanced malignancies, renal carcinoma, pancreatic, gastric and brain tumours. Leading the view that mucin-producing tumours are most often strongly associated with the occurrence of venous thrombosis . HoweverThe pathogenesis of (VG) is obscure; however, venous gangrene does not occur in isolation of venous thromboembolism. Venous gangrene could paradoxically be due to warfarin treatment and develop when the international randomised ratio (INR) is above 6.0, therapeutic range 2.0–3.0). At this supra- therapeutic level of INR the level of protein C is markedly decreased but the thrombin-antithrombin complexes remain unexplainably high .0. At th,9. It haFurthermore, in a study of 158 patients with heparin-induced thrombocytopenia (HIT), 8 patients developed acute venous limb gangrene after heparin therapy was discontinued and warfarin therapy either initiated or continued. In these 8 patients the INR level was at suprat-herpeutic ,11. HIT Antiphosolipid antibodies may also be responsible for the increased venous thrombosis in cancer patients. Anti-phospholipids antibodies are mainly composed of the lupus anticoagulant and anticardiolipin antibodies. These antibodies predispose to thrombosis either by interacting with phospholipids in the platelets and the vascular endothelium or by inhibiting protein C activation and prostacyclin formation in the endothelial cells. It has been reported that the presence of anticardiolipin antibodies appears to double the risk of thrombo-embolic events in cancer patients in comparison with those who are anticardiolipin antibody negative (28% versus 14%) .The hypercoagulable state associated with malignancy is thought to be due to: direct activation of clotting system by cancer cells, and indirectly by activation of platelets, monocyte and endothelial cells. Cytokines such as tumour necrosis factor-α (TNF-α) and interleukin-1β released by tumour cells activate the tissue factor (TF). This transmembrane protein which found only on fibroblasts of vascular adventitia and other stromal cells is also expressed on the surfaces of all solid tumour cells. It initiates coagulation by binding to activated factor VII. Cytokines increase the expression of TF and platelets activating factors (PAF) and decrease the expression of thrombomodulin and the endothelial cells protein C receptors. This imbalance in procoagulant-anticoagulant pathways leads to the hypercoagulable state associated with malignancy leading to the increased risk of thrombosis in cancer patients. In addition, decreased levels of antithrombin III, deficiency of protein C and S were reported with cancer -19. SignImmobilisation, prolonged bed rest, dehydration and vomiting also significantly increase risk of thromo-embolism ,2. In adSeveral studies have described the association between metabolic syndrome and the increased tendency towards hypercoagualtion in general population ,24. The Taking all these factors into consideration, it is possible to postulate that the presence of metabolic syndrome, cancer and paradoxical effect of warfarin may enhance the process of DVT formation and this ultimately may have lead to venous gangrene in cancer patients table .The prevention of venous gangrene is similar to that of VTE. Attention to good nutrition, hydration and early ambulation are of paramount importance in cancer patients table . Use of In contrast, Andtbacka et al, in their retrospective study of 3898 patients with breast cancer, who underwent 4416 surgical procedures for different stages of breast cancer concluded that the risk of VTE following breast cancer surgery is rare (rate of 0.16 per procedure). This lower rate of VTE was achieved by adherence to their clinical pathway using mechanical antithrombotic devices and early ambulation in the postoperative period, without the need for systemic VTE prophylaxis in the form of LMWHs .The conventional treatment of VTE is to start with therapeutic dose of LMWH, followed by warfarin to attain an INR of 2–3 for six months or warfarin for life in cases of recurrent VTE. This regime is very effective in patients without cancer. However, the use of vitamin K antagonist to treat thrombosis in cancer patients is associated with 3-fold increase in the risk of recurrent VTE and up to 6-fold increase in the risk of major bleeding in comparison with non-cancer patients . FurtherA recent systemic review of clinical trails concluded that Low-molecular-weight heparin (LMWH) is modestly superior to unfractionated heparin at preventing recurrent DVT and is at least as effective as unfractionated heparin for treatment of pulmonary embolism . LMWH isIn a case of established VG, warfarin should be stopped and LMWH should be started or continued. It is likely that LMWH provide potential promise as a safe and effective measure in the management of such patients . ImportaVenous gangrene is rare in association with malignancy. The pathogenesis is complex and involves a series of different mechanisms. Hypercoagulable state associated with malignancy and associated increase or decrease in clotting factors, cancer treatment, prolonged immobilisation, surgical operations and metabolic syndrome are all associated with increased risk of VG. The current evidence suggests that LMWH is the drug of choice in the treatment of venous thromboembolism and gangrene. It is safe and effective and associated with lower risk of bleeding, fewer recurrent VTE, lower risk of HIT and venous gangrene in comparison with UFH and Vitamin K antagonists. Further research will reveal the complex mechanisms and provide evidence for safe and effective treatment.


Angiolymphoid hyperplasia with eosinophilia (ALHE) and Kimura's Disease (KD) share many clinical and histopathological features. Although they were once considered different stages of the same disease, they are now known to represent separate entities. Recently, ALHE is being called epithelioid hemangioma (EH), a term that better describes the possible neoplastic nature of the entity.An eighteen year-old Asian female presented with a three-month history of fluctuating swelling and ptosis of the left upper eyelid. Computed tomography disclosed a distinct homogeneous lesion in the left superior orbit, molding to the globe and other orbital structures. At histopathological evaluation the lesion was composed of numerous blood vessels lined by plump endothelial cells with oval nuclei protruding into the lumen. Surrounding the vessels, there was a chronic inflammatory infiltrate with a large proportion of eosinophils. Based on clinical and histopathological findings, the diagnosis of EH was made.Although exams like blood count, urinalysis and whole body scans can assist in the differential diagnosis, EH can be diagnosed and differentiated from KD on histopathological grounds. The presence of vascular hyperplasia with plump endothelial cells protruding into the lumen is the most important discriminator in establishing the diagnosis of EH. Such distinction is crucial for the patient because EH is not associated with any of the systemic manifestations present in KD. Angiolymphoid hyperplasia with eosinophilia (ALHE) and Kimura's Disease (KD) share many clinical and histopathological features.  AlthoughEH usually presents as small, red, pruritic plaques in the subcutis or dermis of the head and neck region. Orbital involvement in EH is a relatively rare manifestation of the disease with only scattered case reports published in literature. An eighteen year-old Asian female presented to the ophthalmology clinic of the McGill University Health Center with a three-month history of fluctuating swelling and ptosis of the left upper eyelid. Mild discomfort was felt whenever the swelling was more intense. A well-defined, soft lesion in the left upper eyelid could be palpated, just below the superior orbital rim, without associated inflammatory signs. No decrease in visual acuity or alterations of extraocular movements was found. Intraocular pressure was 17 mmHg OD and 20 mmHg OS. Computed tomography disclosed a distinct homogeneous lesion in the left superior orbit, molding to the globe and other orbital structures  and Kimura's Disease (KD) share many clinical and histopathological features  AlthoughEH was first described in 1969  It preseKD probably represents an allergic or autoimmune response that typically presents as subcutaneous nodules in the head and neck region of young Asian males  SystemicAlthough exams like blood count, urinalysis and whole body scans can assist in the differential diagnosis, EH can be diagnosed and differentiated from KD on histopathological grounds. The presence of vascular hyperplasia with plump endothelial cells protruding into the lumen is the most important discriminator in establishing the diagnosis of EH. Such distinction is crucial for the patient because EH is not associated with any of the systemic manifestations present in KD.The author(s) declare that they have no competing interests.BF, TH, AAlk and MNBJr were the pathologists that performed the histopathological evaluation. AAlm and BA are ophthalmogists from the Oculoplastics departments and were the attending physician responsible of providing all the clinical information All authors participated in the design of the manuscript. BF, AAlk and AAlm helped to draft the manuscript while TH, BA and MNBJr done the final revisions of the paper. All authors read and approved the final manuscript.


Daphnia populations to recent introductions of non-native fish to assess the relative roles of phenotypic plasticity versus genetic change in causing the observed patterns. The Daphnia community in alpine lakes throughout the Sierra Nevada of California (USA) is ideally suited for investigation of rapid adaptive evolution because there are multiple lakes with and without introduced fish predators. We conducted common-garden experiments involving presence or absence of chemical cues produced by fish and measured morphological and life-history traits in Daphnia melanica populations collected from lakes with contrasting fish stocking histories. The experiment allowed us to assess the  degree of population differentiation due to fish predation and examine  the contribution of adaptive plasticity in the response to predator  introduction.Introductions of non-native species can significantly alter the selective environment for populations of native species, which can respond through phenotypic plasticity or genetic adaptation. We examined phenotypic and genetic responses of D. melanica in response to introduced fish. These phenotypic changes have a genetic basis but are partly due to a direct response to chemical cues from fish via adaptive phenotypic plasticity. Body size showed the largest phenotypic change, on the order of nine phenotypic standard deviations, with approximately 11% of the change explained by adaptive plasticity. Both evolutionary and plastic changes in body size and egg number occurred but no changes in the timing of reproduction were observed.Our results show reductions in egg number and body size of Daphnia populations exposed to chemical cues produced by salmonid fish predators display adaptive plasticity for body size and fecundity. The magnitude of adaptive plasticity was insufficient to explain the total phenotypic change, so the realized change in phenotypic means in populations exposed to introduced fish may be the result of a combination of initial plasticity and subsequent genetic adaptation. Our results suggest that immediately following the introduction of fish predators, adaptive plasticity may reduce the impact of selection through "Baldwin/Bogert effects" by facilitating the movement of populations toward new fitness optima. Our study of the response of a native species to an introduced predator enhances our understanding of the conditions necessary for rapid adaptive evolution and the relationship between rapid evolution and adaptive phenotypic plasticity.Native  Introductions of non-native species can result in strong selective challenges for native populations. The strength of selection in this case is determined by the size of the environmental shift, which imposes a fitness cost on the population proportional to the squared distance between the population mean phenotype and the position of the new optimum ,2. If thi.e., variance that is context-dependent and arises only in response to specific environmental cues) and changes in covariances between traits that increase the response to selection , thus, an increase of one individual at first reproduction might substantially offset any losses due to predation. An interesting aspect of our findings is that our naive Daphnia populations, those without any history of fish exposure, are responsive to chemicals produced by fish. This observation suggests that D. melanica may be pre-adapted to fish predation, and that the genetic machinery responsible for adaptive phenotypic plasticity in response to fish kairomone is ancestral in this species.Our results suggest that naive  effect" ,8. Fish  effect" ,39-41. TAlthough we find evidence for adaptive phenotypic plasticity that would facilitate short-term population persistence in the face of novel predation, it is not sufficient to explain the difference in body-size and egg number between populations that are historically fishless and those that co-occur with fish. For example, a comparison of the average body size in the Evelyn Lake population (1.34 mm in the kairomone(-) and 1.24 mm in the kairomone(+) treatments) with the average in the fishless populations (2.00 mm in the kairomone(-) and 1.95 mm in the kairomone(+) treatments) shows that the mean phenotype in Evelyn Lake has diverged by 9.4 phenotypic standard deviations. The change in body size attributable to plasticity in fishless populations is approximately one standard deviation. In other words, the change in body size due to plasticity accounts for only about 11% of the total difference observed between Evelyn and fishless populations. Thus, the phenotypic differences observed in our study appear largely due to changes in the underlying genetic components controlling phenotype.Our observation that the body-size response in Evelyn Lake was much higher than that in Puppet Lake could arise for three reasons. First, the difference in body size could simply reflect the different amounts of time each population was exposed to fish predation . Second, Daphnia populations may have experienced differing levels of fish predation resulting in varying selection intensities, with the selection intensity in Evelyn Lake substantially higher. Finally, given our observation that expressed levels of genetic variance for body size increased approximately 6-fold in response to fish kairomone in one fishless population (Frog Lake) but not in the other (Source Lake) our fish populations may have differed in the initial levels of standing genetic variation, with the Evelyn Lake population harboring more standing genetic variation than Puppet Lake.D. melanica to deposit large amounts of melanin in the carapace. Melanin production in the carapace would initially "blind" selection to changes in egg number. Thus, an evolutionary response in egg number should occur only after reductions in melanin deposition. Fish predation produces strong selection on melanin production in other Daphnia populations . Therefore, the apparent delayed onset of selection on egg number could be due to initial selection on melanin production and subsequent selection on egg number.Our observation that Evelyn Lake was the only population to display a significant evolutionary reduction in egg number is likely due to the ability of ulations , and theDaphnia suggest adaptive evolutionary changes in one trait are not necessarily associated with concomitant maladaptive changes in others . The moInterpretation of results based on our model is relatively straightforward. A significant treatment effect is evidence for phenotypic plasticity in a given trait, irrespective of a populations' fish stocking history. A significant lake type effect implies phenotypic differences between populations in historically fishless and fish-containing lakes. The strength of conclusions about the actual level of genetic differentiation underlying phenotypic divergence is based on the level of significance of the interaction term. For example, a significant effect of lake type in conjunction with non-significant interaction terms would indicate underlying genetic differences among populations regardless of treatment effects.Plasticity in the expressed genetic variance of traits was assessed by calculating coefficients of variation (CV) for each population across treatments separately. We then constructed 95% Modified McKay confidence intervals for each CV ,55 and aDaphnia populations, assisted with fieldwork in the Sierra Nevada, and helped draft the manuscript. MEP conceived of the study, conducted fieldwork in the Sierra Nevada to collect the experimental populations, and contributed to analysis and manuscript preparation. All authors read and approved the final manuscript.LCL designed and conducted the experiment, performed statistical analyses, and drafted the manuscript. JWB conducted the experiment. RAK provided background information on


Traditionally metastatic melanoma of the distal leg and the foot metastasize to the lymph nodes of the groin. Sometimes the first site of nodal disease can be the popliteal fossa. This is an infrequent event, with rare reports in literature and when it occurs, radical popliteal node dissection must be performed.We report a case of a 36-year old man presented with diagnosis of 2 mm thick, Clark's level II-III, non ulcerated melanoma of the left heel, which developed during the course of the disease popliteal node metastases, after a superficial and deep groin dissection for inguinal node involvement. Five months after popliteal lymph node dissection he developed systemic disease, therefore he received nine cycles of dacarbazine plus fotemustine. To date (56 months after prior surgery and 11 months after chemotherapy) he is alive with no evidence of disease.In case of groin metastases from melanoma of distal lower extremities, clinical and ultrasound examination of ipsilateral popliteal fossa is essential. When metastatic disease is found, radical popliteal dissection is the standard of care. Therefore knowledge of anatomy and surgical technique about popliteal lymphadenectomy are required to make preservation of structures that if injured, can produce a permanent, considerable disability. Involvement of the regional lymph nodes is the most important predictor of outcome in patients with cutaneous melanoma . Traditi2 plus dacarbazine 900 mg/m2 intravenously, every three weeks, for a total of nine cycles) and in September 2006 the patient is alive with no evidence of disease . Preoperative lymphoscintigraphy showed uptake of the radiotracer in three nodes of ipsilateral groin that after removal resulted on final pathology negative for metastatic melanoma. In the February 2005 the patient presented at follow-up with clinical nodal disease in the left groin confirmed by fine needle aspiration cytology (FNAC). Preoperative staging did not reveal signs of systemic disease, therefore the patient underwent an inguinal lymph node dissection, that revealed 1 positive lymph node out of 8, and concomitant iliac and obturator lymph node dissection, with 3 positive nodes out of 11. Three months later he developed a mild-grade lymphedema of the inferior left extremity. Ultrasounds study (US) and magnetic resonance imaging (MRI)  were recorded as having developed metastatic disease in a popliteal node. Five of 13 patients with popliteal disease had metastatic inguinal node at the time or before the presentation of popliteal disease. Eight of the 13 patients with popliteal node metastases subsequently developed distant metastatic disease, at mean interval of 39 months (range 3–127 months) from the time of treatment of their primary melanomas. At last follow-up reported  seven of the patients had died of melanoma and six were alive, all clinically disease free. Thompson demonstrated that if a node in the popliteal fossa is observed on lymphoscintigraphy, a sentinel lymph node biopsy is indicated. If the specimen is positive for melanoma, a popliteal node dissection is the logical next step. So they concluded that there are two indications for popliteal node clearance: a histologically positive sentinel node in the popliteal fossa or clinical evidence of metastatic disease in this area [et al., recognized 106 patients with melanoma at the knee or distally. Ten patients (9%) had drainage to the popliteal basin at lymphoscintigraphy. They identified a 2.8% rate of metastatically involved popliteal nodes. Metastasis was detected in one patient by immunohistochemical analysis and were palpable in two at presentation. All had concomitant drainage to the groin, evident clinically or by lymphoscintigraphy. In the patient, with positive popliteal sentinel lymph node, groin sentinel node was disease free, so he underwent radical popliteal dissection. Two patients, with synchronous palpable popliteal and groin metastases, underwent radical popliteal and groin dissection. All 3 patients relapsed a short time after surgery with systemic and in-transit metastases; all 3 subsequently died. The authors concluded that popliteal area should always be examined when the primary lesion involves the distal lower extremities and when metastatic disease is found, radical popliteal dissection is the standard of care [Thompson  of care .In the extremities drainage of cutaneous melanoma is considered more predictable than in the head and neck . Usuallyet al., showed that positive nodes in the popliteal fossa can be expected from a lesion located anywhere below the knee [Previous authors have suggested that sites which drain to the popliteal nodal basin are located on the posterolateral aspect of the heel, below the lateral malleolus. However, Thompson the knee .et al., considered that in case of patients with popliteal involvement having metastatic inguinal nodes at the time or before the presentation of popliteal disease, popliteal metastasis could represent "backflow" from inguinal metastasis or in transit metastasis [In our case primary site was at the postero lateral aspect of the left heel. At presentation there was no evidence of clinical disease in the ipsilateral groin or popliteal fossa. SLNB showed three negative nodes. Recurrence after tumour-negative SLNB may result from an error in the technique of sentinel node detection at the time of lymphoscintigraphy or the operation, an inaccurate histopathological stain, or the biologic features of the melanoma . In our tastasis .The utilitarian surgical approach to the popliteal fossa involves a Z-plasty incision, to obtain optimal exposure of diamond shaped popliteal space. The initial step of popliteal dissection is adequate exposure of the popliteal fossa and identification of the neurovascular bundle. Identification of the popliteal fascia, which is very thin and friable, is a basic surgical step. This structure serves as a landmark because of its close proximity to the neurovascular bundle. As the fascia is exposed, the most superficial key anatomic structures are the lesser saphenous vein, that must be ligated and divided, and some small cutaneous nerve terminal branches. Next the incision of the deep fascia must be careful because of the superficiality of the nerves. The tibial nerve is the most superficial structure within the popliteal fossa  likewise the common peroneal nerve. A fat pad overlying and alongside the popliteal vessels is reported to enclose two to seven lymph nodes . With caThe case described was peculiar in the time of appearance of popliteal and groin metastases. Although the clinical evidence of groin involvement come as first site of recurrence, it must be considered as a consequence of popliteal metastases probably present since the sentinel node biopsy. In all cases of groin metastases from melanoma of distal lower extremities, clinical and ultrasound examination of ipsilateral popliteal fossa is essential, especially where the foot is the most frequent site of malignant melanoma, like in Japan . When meThe author(s) declare that they have no competing interests.UM conceived the study, carried out the literature search, and draft the manuscript; CC helped in management of the patient and preparation of the manuscript; MGC and GB carried out literature review and manuscript drafting; NM shaped the idea for the manuscript, coordinated the study and edited the manuscript.All authors read and approved the final manuscript.


The brain is deemed “immunologically privileged” due to sparse professional antigen-presenting cells and lymphatic drainage, and to the blood-brain barrier. Although the actual extent of this privilege is controversial, there is general consensus about the limited need in intracerebral neural grafts for immunosuppressive regimens comparable to those used in other cases of allotransplantation. This has led over the past fifteen years to the use of either short-term or even no immunosuppression in most clinical trials with foetal neural transplant in patients with Parkinson's and Huntington's disease.We report biological demonstration of alloimmunisation without signs of rejection in four grafted patients out of 13 studied during the course of a clinical trial involving fetal neural transplantation in patients with Huntington's Disease. Biological, radiological and clinical demonstration of an ongoing rejection process was observed in a fifth transplanted patient. The rejection process was, however, fully reversible under immunosuppressive treatment and graft activity recovered within six months.There had been, up to date, no report of documented cases that could have cast a doubt on those procedures. Our results underline the need for a reconsideration of the extent of the so-called immune privilege of the brain and of the follow-up protocols of patients with intracerebral grafts. It also suggests that some of the results obtained in past studies with foetal neural transplants may have been biased by an unrecognized immune response to donor cells. Transplantation of cells and tissues to the mammalian brain and CNS has been clinically assessed for more than 15 years, in several hundred patients with Parkinson or Huntington Disease. Because the brain was considered differently than almost all other organs, in terms of the possibility of an immune response, little consideration has been given up to now either to the potential existence of adverse consequences of a rejection process, or even to the existence any sign of allo-immunisation in patients. The mechanisms underlying this privilege are diverse, and probably cumulative, and results from the blood– brain barrier, the absence of professional antigen-presenting cells in the brain and the sparse lymphatic drainage from the central nervous system  is currently ongoing in Europe, following the promising results of a pilot study This protocol involves a temporary immunosuppressive therapy with cyclosporin A stopped during the first year after bilateral grafting, azathioprin and prednisolone progressively decreased then stopped over the following months. The present study has been organized as an ancillary to the main trial. It concerned 13 patients , who hadAssessment protocols and imaging methods have been described in referenced studies Fourteen months after grafting, one patient (patient 3) started to exhibit general alteration of clinical status with weight loss of 22 kg over three months, worsened choreic movements lateralised to the left side, gait disorders and falls. Brain MRI showed an ongoing encephalitic process with vasogenic oedema, extending from the striatum to the frontal cortex mostly on the right side . PositroAfter ruling out an infection on the basis of the lack of alteration of blood cells count, a graft rejection was hypothesized. A humoral response was indeed documented with anti-HLA I –but not II– antibodies in the blood  and in the cerebrospinal fluid . The antibodies (IgG) were specifically directed against A3 and A11 class I HLAs. Control assay in a serum sample frozen immediately before surgery was negative for HLA antibodies, indicating post-operative occurrence of the specific B lymphocyte response. Biological demonstration of alloimmunisation against the graft tissues was provided by haplotyping preserved frozen samples from the three foetuses used for grafting, with HLA-A*3 allele in the foetus grafted on the right side, HLA-A*11 in one of the two foetuses grafted in the left striatum.Aggressive acute anti-inflammatory treatment associated to reinstatement of the original immunosuppressive regimen rapidly led to regression of MRI signs of encephalitis. General clinical status remained altered for 4 more months then recovered, the patient regaining more than half of her lost weight. Anti-HLA I antibodies disappeared from blood and CSF samples. After 7 months of treatment, chorea lessened back to pre-rejection levels, while gait disorders and falls disappeared. The patient regained more than half of the lost weight. PET scan imaging then revealed the presence in the right striatum of two areas of increased metabolic activity  as compaAlloimmunisation to donors' antigens was sought in 12 other MIG-HD grafted patients followed up different times beyond the end of the period of immunosuppressive treatment. Four of them demonstrated alloimmunisation to one of the donors although none displayed either clinical or radiological signs of an ongoing rejection process Click here for additional data file.


Palindromes are known to be involved in a variety of biological processes. In the present investigation we carried out a comprehensive analysis of palindromes in the mitochondrial control regions (CRs) of several animal groups to study their frequency, distribution and architecture to gain insights into the origin of replication of mtDNA.Many species of Arthropoda, Nematoda, Mollusca and Annelida harbor palindromes and inverted repeats (IRs) in their CRs. Lower animals like cnidarians and higher animal groups like chordates are almost devoid of palindromes and IRs. The study revealed that palindrome occurrence is positively correlated with the AT content of CRs, and that IRs are likely to give rise to longer palindromes.The present study attempts to explain possible reasons and gives in silico evidence for absence of palindromes and IRs from CR of vertebrate mtDNA and acquisition and retention of the same in insects. Study of CRs of different animal phyla uncovered unique architecture of this locus, be it high abundance of long palindromes and IRs in CRs of Insecta and Nematoda, or short IRs of 10–20 nucleotides with a spacer region of 12–14 bases in subphylum Chelicerata, or nearly complete of absence of any long palindromes and IRs in Vertebrata, Cnidaria and Echinodermata. Escherichia coliA DNA palindrome is a unique case of inverted repeats (IRs) Metazoan mitochondrial DNA (mtDNA) is a closed-circular, double-stranded molecule, ranging in size from 15 to 20 kb From NCBI, sequences of mitochondrial CRs were queried in 12 most studied animal phyla  and were downloaded from different phyla/classes/orders separately wherever necessary, by carrying out a boolean search using combination of different keywords. For example, to download the CR sequences of lepidopteran species, we used keywords like lepidoptera AND control region or lepidoptera AND D loop. Each sequence description was manually checked to ensure that we downloaded CR sequences only.To study the abundance of palindromes and IRs in subphyla, classes and orders of the phyla Arthropoda and Chordata, CR sequences were downloaded separately from three subphyla Chelicerata, Crustacea and Uniramia of phylum Arthropoda. Subphylum Uniramia was further divided into Chilopoda and Insecta. Many CR sequences have been reported in class Insecta. Therefore, we further binned Insecta CRs based on the taxonomic order of origin. From phylum Chordata we extracted CRs from two important subphyla, Cephalochordata and Vertebrata. Details of the sample size are given in Finally, based on number of sequences available in each phylum we divided the data into eight animal groups namely Cnidaria, Nematoda, Mollusca, Echinodermata, Chelicerata, Crustacea, Insecta and Vertebrata , two fish species  and a reptile (Teratoscincus keyserlingii). IRs were found only in one species, T. keyserlingii out of 125 vertebrates analysed.In vertebrate mitochondrial CRs analysed in the present study, palindromes were found in a few avians , of 53 species analysed in this order, harbored palindromes. When compared to order Diptera, lepidopterans possessed higher number of palindromes in CRs. Out of 50 dipterans, 38 had palindromes  of the 53 lepidopterans studied. Even though palindromes are unstable due to several reasons listed above, they are retained in CR suggesting their possible involvement in replication initiation of lepidopteran mitochondrial genome. However, in dipterans we observed shorter palindromes of only up to 56 bp. None of the CR sequences analysed in the remaining phyla possessed palindromes greater than 150 bp. However, molecular function of these long palindromes in insect mtDNA needs further analysis.Although palindromes exist naturally in the DNA sequence of many organisms, it is difficult to maintain long palindromes because of their genetic instability stability as demonstrated in Since insect CRs are AT-rich, it prompted us to establish correlation if any between AT content and palindrome occurrence. All CRs were pooled irrespective of the phyla to which the species belonged and analysed for presence or absence and number of palindromes in relation to AT content. Positive correlation between AT content and number of palindromes with correlation co-efficient of 0.89 was observed. Through these analyses we propose that palindromes originate in CRs with more than 85% AT content .All animal groups except Mollusca and Echinodermata differed significantly (p<0.05) in their CR AT content . CnidariAnalysis of complete mitochondrial genomes of Insecta and Nematoda also indicated the AT richness in their genomes . NematodThe present study revealed surprisingly long palindromes in mitochondrial origin of replication. It is known that palindromes and IRs reside near the origin of replication in several bacteria, plasmids and viruses E. coliE. coliE. coli. The presence of a fewer IRs led us to suspect that intervening regions between IRs would be deleted over a period of time resulting in formation of long palindromes. This can be explained in the following ways: i. when the DNA melts either during DNA replication or due to any other cellular activity, there would be intra-strand base-pairing in the regions of IRs leading to formation of hair-pin loop. The unpaired spacer regions between IRs may be eventually cleaved by DNases, as the tips of hairpin loops are sensitive to single-stranded nucleases Long palindromic sequences are unstable since they are deleted at extremely high rates as reported in the case of However, we cannot exclude other factors, which are thought to lead to formation of long palindromes in CRs of insects, as in some cases they form as a result of a precisely regulated developmental program. For example, previous reports have shown the formation of long palindromes from short IRs by single strand annealing of IRs followed by removal of nonhomologous DNA and gap-filling DNA replication Palindromes of 4–6 bp are implicated in bacterial chromosome and plasmid replication. Since mitochondria are thought to have originated from bacteria, the origin of replication also has a propensity to harbor palindromes. But palindromes present in CRs of insects are much longer than those present in bacterial origin of replication. Phylogenetic evidence derived from rRNA Due to varied evolutionary forces acting on different species, lower animal groups like Cnidaria and higher animal groups like echinodermates may be completely devoid of palindromes and, longer palindromes would have originated or expanded from already present smaller palindromes or IRs in some species of insects and nematodes, depending on the bio-physiochemical environment within the cell. Although the genetic role of mtDNA appears to be universally conserved, this genome exhibits remarkable variation in conformation and size, as well as in actual gene content, arrangement and expression The surprising absence of long palindromes and IRs in CR of vertebrate mtDNA implies that during the course of evolution, higher animals like chordates probably have adopted a slightly different mechanism of replication of mtDNA which does not require palindromes as a recognition motif in replication initiation. The variation in distribution of palindromes and IRs in different animal groups suggests that the mode of replication origin is quite different between different animal phyla. This is supported by the previous studies on CR of vertebrates and insects The AT bias is generally observed in insect mitochondrial genomes, which ranges from 69.5 to 84.9% Gallus sps  and DNA synthesis proceeds unidirectionally. When the synthesis of H-strand reaches two-thirds of the genome, the synthesis of L-strand (OL) is initiated from the replication origin of the L-strand located two-thirds of the genomic distance away from the replication origin of H-strand Metazoan mtDNA contains a distinct replication origin on each of the DNA strands. The position of the replication origin and mode of replication have been studied in detail in mammalian mtDNA and also recently in insects H and are suggested to be implicated in the initiation of the H-strand replication L, the IR sequence of 10–12 bp, that could form a stem-loop configuration is conserved among vertebrate species and is also proposed to be required for the initiation of replication In vitro replication studies have suggested that the IR sequence serves as a recognition motif for mtDNA primase which provides a short RNA primer for L-strand synthesis, and DNA synthesis is initiated near the base of the stem-loop structure utilizing the 3′ ends of the RNA primer Several regulatory sequences have been identified in the CR of the vertebrate mtDNA. These are present immediately upstream of the ON) is located in the middle portion of CR. Synthesis of minor coding strand proceeds unidirectionally, and the major coding strand synthesis begins after 97% of the minor coding strand synthesis is completed J) must be located 97% of the genomic distance away from the replication origin for minor strand, that is, within the CR H in vertebrates N and OJ in insects In insects, the leading and lagging strands are termed minor and major coding strands according to the relative numbers of the gene encoded on the respective DNA strand J is located 97% genomic distance away from the ON that is, both OJ and ON lie within the CR. This observation is consistent with the recent findings in Drosophila mtDNA that the free 5′ ends in the CR near the tRNAIle gene, exactly 97% genomic distance away from the ONJ of insects and OL of vertebrates, there should be presence of IRs in OJ like in OL to form stem loop structure for the initiation of replication from OJ. Indeed in the present study we have found palindromes and IRs in CRs of insects.According to the strand-asynchronous, asymmetric model of vertebrate mtDNA, the replication of the L-strand is initiated when the synthesis of the H-strand passes beyond the L-strand origin, and template strand for the L-strand replication becomes single stranded. In insects OThis is a comprehensive report on the analysis of palindromes and IRs comprising 393 CRs from seven animal phyla. We are reporting that long palindromes and IRs are abundant in insect mitochondrial origin of replication. Lower animals like cnidarians and higher animal groups like chordates are almost devoid of long palindromes, where as many species of Arthropoda, Nematoda, Mollusca and Annelida harbor palindromes and IRs in their CRs . Here weSupplementary File S1Details of the control region sequences used in the present study(0.10 MB XLS)Click here for additional data file.Supplementary File S2P-values obtained by t-test performed on AT percent values of 8 animal groups.(0.01 MB PDF)Click here for additional data file.Supplementary File S3CLUSTAL X - Multiple sequence alignment of short palindromes found in vertebrates(1.61 MB PDF)Click here for additional data file.