Neuroendocrine tumours frequently metastasize to the liver. Although generally slowly progressing, hepatic metastases are the major cause of carcinoid syndrome and ultimately lead to liver dysfunction, cardiac insufficiency and finally death.A literature review was performed to define the optimal treatment strategy and work-up in patients with neuroendocrine hepatic metastases. Based on this, an algorithm for the management of these patients was established.131I-MIBG treatment or targeted chemo- and radiotherapy should be attempted. A final option for selective patients is orthotopic liver transplantation.Platelet serotonin and chromogranin A are useful biomarkers for detection and follow-up of neuroendocrine tumour. Helical computed tomography and somatostatin receptor scintigraphy are the most sensitive diagnostic modalities. Surgical debulking is an accepted approach for reducing hormonal symptoms and to establish better conditions for medical treatment, but is frequently impossible due to the extent of disease. A novel approach is the local ablation of tumour by thermal coagulation using therapies such as radiofrequency ablation (RFA) or laser induced thermotherapy (LITT). These techniques preserve normal liver tissue. There is a tendency to destroy metastases early in the course of disease, thereby postponing or eliminating the surgically untreatable stage. This can be combined with postoperative radioactive octreotide to eliminate small multiple metastases. In patients with extensive metastases who are not suitable for local destruction, systemic therapy by octreotide, Treatment for patients with neuroendocrine hepatic metastases must be tailored for each individual patient. When local ablative therapies are used early in the course of the disease, the occurrence of carcinoid syndrome with end stage hepatic disease can be postponed or prevented. Carcinoids are neuroendocrine tumours that arise from neoplastic proliferation of enterochromaffin or Kulchitsky cells . In 1963Neuroendocrine hepatic metastases represent about 10% of all hepatic metastatic neoplasm's . These mNeuroendocrine tumours of the small intestine produce large quantities of serotonin (5-hydroxytryptamine), reflected in raised levels of platelet serotonin and a high urinary excretion of 5-hydroxyindoleacetic acid (5-HIAA) ,12. The Plasma chromogranin A (CgA) has been claimed the most sensitive and specific marker of tumour volume . CgA is Both tumour markers, platelet serotonin and CgA, can be reliably used for diagnosis of neuroendocrine tumour and for monitoring the outcome of treatment in individual patients.Several imaging modalities are available to detect hepatic metastases and their primary neuroendocrine tumours. Conventional ultrasonography (US), computed tomography (CT), magnetic resonance imaging (MRI) and somatostatin receptor scintigraphy (SRS) are the cornerstones for the localisation of neuroendocrine tumours with sensitivities of respectively 46%, 42%, 43% and 90% -23. The Surgical resection is to be considered when no extrahepatic disease is present. Hemihepatectomy or segmental resection is feasible when metastases are solitary and resection can be radical with enough functional liver tissue remaining. Symptomatic response rates have been reported to be 90% for a mean duration of 19.3 months after surgical resection . UnfortuLocal therapy using radiofrequency ablation (RFA) or laser induced thermotherapy (LITT) is a well-established treatment for unresectable hepatocellular carcinomas and liver metastases from colorectal carcinomas ,28. A feAll invasive procedures during surgery such as liver resection and ablation and even anaesthesia can induce hormone release and even provoke a life-threatening carcinoid crisis . In the Hepatic arterial embolisation with or without chemotherapy is a palliative option for those unresectable lesions in which the predominant mass of tumour is localised in one of the liver lobes. In the past, more radical blunt techniques to occlude the main hepatic artery were used. However, recently, superselective techniques have become available with the advantage of leaving the main segmental arteries open. Contraindications of hepatic arterial embolisation include complete portal vein occlusion, hepatic failure and previous biliary anastomoses . Symptom131I-MIBG), interferon-α (IFN-α) or targeted chemo- and radiotherapy. Octreotide is a somatostatin analogue and appears to be an efficacious treatment for carcinoid syndrome, reducing symptoms in more than 70% of patients [131I-MIBG therapy. In individual cases, improved quality of life may be obtained [131I-MIBG treatment [90Y and 177Lu. Experimental studies in patients who have somatostatin-positive tumours show complete remission by the use of tetra-azacyclododecane tetra-acetic acid Tyr3-octreotide [90Y, Tyr-octreotide, 177Lu or 131I-MIBG treatment appears to be a reliable and well-tolerated radionuclide therapy and might be a useful adjunct in patients with malignant neuroendocrine carcinoma, providing long-lasting palliation and prolonged survival [Pharmacological therapy consists of long-lasting octreotide injections, Iodine-131 metaiodobenzylguanidine  declare that they have no competing interests.LV reviewed the literature and drafted the manuscript. IBR and CL critically reviewed the paper and were involved in the preparation of the final manuscript. RH was involved in the conception of the work and manuscript preparation. All authors read and approved final version for publication.


Part II of this review examines the functional "short leg" or unloaded leg length alignment asymmetry, including the relationship between an anatomic and functional leg-length inequality. Based on the reviewed evidence, an outline for clinical decision making regarding functional and anatomic leg-length inequality will be provided.Online databases: Medline, CINAHL and Mantis. Plus library searches for the time frame of 1970–2005 were done using the term "leg-length inequality".The evidence suggests that an unloaded leg-length asymmetry is a different phenomenon than an anatomic leg-length inequality, and may be due to suprapelvic muscle hypertonicity. Anatomic leg-length inequality and unloaded functional or leg-length alignment asymmetry may interact in a loaded (standing) posture, but not in an unloaded (prone/supine) posture.The unloaded, functional leg-length alignment asymmetry is a likely phenomenon, although more research regarding reliability of the measurement procedure and validity relative to spinal dysfunction is needed. Functional leg-length alignment asymmetry should be eliminated before any necessary treatment of anatomic LLI. In Part I of this review, the literature regarding the prevalence, magnitude, effects and clinical significance of anatomic leg-length inequality (LLI) was examined. Using data on leg-length inequality obtained by accurate and reliable x-ray methods, the prevalence of anatomic inequality was found to be 90%; the mean was 5.2 mm (SD 4.1). The evidence suggested that, for most people, anatomic leg-length inequality is not clinically significant until the magnitude reaches ~20 mm (~3/4"). The phenomenon of the functional "short leg" will be considered in Part II of this review. The objective is to define functional "short leg", how it differs from anatomic LLI and explore any association with neuromuscular dysfunction. In addition we will review the apparent efficacy of heel lifts in some cases of mild anatomic LLI, plus muscular reactions to, and causes of, pelvic torsion.The functional short leg, or unloaded leg-length alignment asymmetry (hereafter abbreviated as LLAA) is itself a phenomenon much discussed and little understood. Essentially, when a subject lies prone or supine, unloading the pelvis, the feet are examined, most often at the welt (heel-sole interface), for the presence of a "short leg" or alignment asymmetry. Some hold the opinion that anatomic LLI can be measured in this way . The exaAnatomic LLI is caused by a natural developmental asymmetry or a variety of other factors, including fracture, disease, and complications of hip replacement surgery. Given the long-term loading, the lumbopelvic structure may be expected to adapt via Heuter Volkmanns' law  and softUsing a device to measure standing pelvic crest unleveling, Petrone et al found excellent intra and inter-examiner reliability, and validity ICC, 0.89–0.90) relative to anatomic leg length inequality determined by x-ray measurement in asymptomatic subjects [9–0.90 reafter they were skeletally mature [Lumbar lateral flexion was studied in a group of subjects 10 years after LLI caused by femoral fracture that occurred y mature . Despitey mature . Signifiprior to skeletal maturity [However, another study from the same orthopedic center looked at the effects of significant (mean 3 cm) LLI acquired maturity  in now mThis type of permanent compensation to pre skeletal maturity LLI was also found in subjects with pelvic unleveling. Young et al  found thThese two studies ,11 proviThe persistence of pelvic torsion in subjects with anatomic LLI is supported by Klein  who founThe studies noted above provide indirect evidence that the pelvic torsion associated with childhood-onset anatomic leg-length inequality is adapted for and incorporated as normal. It follows then, that when an average person with an anatomic LLI and structurally compensatory pelvic torsion moves from a loaded (standing) to an unloaded (prone/supine) position, the torsion of the pelvis remains intact and the leg length at the feet/shoes would appear "even" on a visual check. The pelvis – joints, ligaments and muscles – have adapted to the anatomic LLI, making any torsion structural. It is this putative biomechanical adaptation that makes unloaded leg-length alignment asymmetry tests – the functional "short leg" tests – unreliable as a measure of anatomic LLI .Unloaded LLAA is suspected to result from hypertonicity of suprapelvic muscles -17. In aWhen standing, the actions of the QL depend on whether the spine or the pelvis is stabilized. If the pelvis is stabilized, QL contraction laterally flexes and extends the spine ,20,21. WNow we can return to the dilemma of how lifts may have a positive effect on back pain and muscle activity given that most anatomic LLI is not clinically significant. Torsion of the pelvis as an adaptive structural compensation in anatomic LLI has been shown to be limited. If a person has pelvic torsion due to anatomic LLI near the limits of the body's ability to adapt, and QL hypertonicity with its ability to cause pelvic torsion is superimposed, muscular bracing reactions and pain could be the result. Indahl et al  found thBased on their research, Allum et al  proposedIn a case of additive effects of anatomic LLI and QL/suprapelvic hypertonicity on pelvic torsion, a lift used to level the pelvis would take the strain off the sacroiliac and associated joints and ligaments and decrease potentially painful muscular bracing. Thus, lifts can work to decrease back pain in people with what seem to be clinically insignificant amounts of anatomic leg-length inequality. Of course, it would be important for the clinician to explore reasons for any quadratus lumborum and other suprapelvic muscle hypertonicity and eliminate them to provide a complete correction. On the other hand, pure anatomic LLI in the range of and above 20 mm – the upward limit for adaptive compensation – may stimulate sacroiliac and/or lumbar proprioceptors causing reflexive and ultimately painful muscular contractions that will only be relieved by a lift to level the pelvis.Reliable detection of LLI and LLAA is difficult, but not impossible. Research has shown the examination procedures for putative LLAA both prone  and supiIn addition to reliability, the leg check procedure outlined by Cooperstein et al demonstrated concurrent validity as assessed against artificial LLI . HoweverOnce any suprapelvic muscle hypertonicity has been relieved – and the causes may be multiple, including upper cervical joint dysfunction ,29-33 – Anatomic leg-length inequality under 20 mm and leg-length alignment asymmetry caused by supra-pelvic muscle hypertonicity may interact in a loaded (standing) posture, but not in an unloaded (prone/supine) posture. Any leg-length alignment asymmetry due to suprapelvic muscular hypertonicity should be eliminated before any necessary treatment of anatomic leg-length inequality. By using this information, which is open to change based on new studies, the clinician may better understand the diverse and sometimes confusing findings relative to anatomic leg-length inequality and functional or unloaded leg-length alignment asymmetry, and be better able to make treatment recommendations.The author(s) declare that they have no competing interests.


The authors discuss a study in PLoS Biology that investigated whether the lower levels of HDL cholesterol in patients with HIV may be due to impaired cholesterol efflux from macrophages. Cardiovascular disease is an increasing cause of morbidity in HIV-infected adults receiving antiretroviral therapy (ART). ART, particularly protease inhibitors and to a lesser extent nucleoside analogue reverse transcriptase inhibitors, can adversely affect lipid and glucose metabolism . MoreoveHDL cholesterol levels are reduced in untreated HIV infection  and in hPLoS Biology, Mujawar et al. investigated whether these lower levels might be due to impaired cholesterol efflux from macrophages [ABCA1 cause Tangier disease, which is associated with low HDL cholesterol and accelerated atherosclerosis [The pathogenesis of low HDL cholesterol levels in untreated HIV infection is unknown. In a study recently published in rophages , a proceclerosis .nef-transfected cells exhibited increased apoA-I binding, apoA-I internalization was blocked, suggesting that nef at the plasma membrane may prevent ABCA1 internalization and subsequent apoA-I lipidation. As cholesterol is required for HIV replication [The researchers found that expression of HIV nef, a protein that enhances HIV replication and infectivity, specifically inhibited ABCA1-dependent cholesterol efflux from macrophages (the precursors of foam cells in atherosclerotic plaque) and re-localized ABCA1 to an exclusive plasma membrane distribution. While lication , MujawarThere is a strong correlation between ART duration and the risk of myocardial infarction.Toxoplasma gondii, in which endocytosed low-density lipoprotein cholesterol is diverted to a specialized vacuole to support parasite growth and replication [Mujawar et al. propose that HIV nef redirects cholesterol from an ABCA1-mediated efflux pathway to virus-controlled cholesterol transport in order to ensure sufficient cholesterol for virion assembly. The interruption of a host cholesterol trafficking pathway by an intracellular pathogen is also employed by lication . Given tlication , the finIt remains to be determined, however, if this mechanism is the greatest contributor to these low levels. If so, one would expect that HDL cholesterol levels would fall rapidly after primary HIV infection and return to pre-infection levels with effective ART that did not affect lipid metabolism. In addition, the observed reductions might be inversely proportionate to plasma HIV RNA levels. Alternatively, low HDL cholesterol levels may also be caused by ART-induced lipodystrophy. This ART side effect is characterized by peripheral lipoatrophy and relative central adiposity and is strongly associated with low HDL cholesterol levels as well as insulin resistance and hypertriglyceridemia, all features of congenital lipodystrophies in which abnormalities of ABCA1 have not been identified ,15,16.ABCA1, that could attenuate virion-associated cholesterol? Treatment of HIV-infected ABCA1-deficient macrophages with LXR ligands should establish a definitive role for ABCA1 in countering HIV replication.The findings of Mujawar et al. raise several important questions. Is the apparent nef-stimulated decrease in ABCA1 protein levels due to ABCA1 degradation, or possibly due to altered partitioning of ABCA1 into detergent-resistant lipid domains? Does nef physically interact with ABCA1? Use of the myristoylation-deficient nef in this study only suggests that nef must be targeted to the plasma membrane—likely to cholesterol rafts . FurtherThe present data provide a clue as to why HIV infection, as well as ART, might accelerate atherosclerosis. The data support the shift away from a paradigm of delaying or stopping ART to reduce the risk of cardiovascular disease and raise the possibility that antiretroviral drugs without direct metabolic effects may actually reduce cardiovascular risk. The data also emphasize the need for clinicians to consider all the metabolic effects  of each antiretroviral drug, not just total cholesterol levels, in the management of cardiovascular risk in HIV-infected adults.The study by Mujawar et al. also has important implications for developing new approaches for suppression of HIV replication. Selective LXR modulators under active development for treatment of atherosclerosis may prove useful as ART.The long-term effects of each antiretroviral drug on HDL cholesterol, as well as other lipid and glycemic parameters, need to be more completely analyzed in randomized trials, as well as in shorter healthy volunteer studies. It remains to be determined whether new antiretroviral drug classes, such as HIV fusion, CCR5, and integrase inhibitors, will be free of these metabolic complications.


An analysis of humans and mouse genomes indicates that gene function, expression level, and sequence conservation influence transposable elements insertion/fixation in mammalian introns. Transposable elements (TEs) represent more than 45% of the human and mouse genomes. Both parasitic and mutualistic features have been shown to apply to the host-TE relationship but a comprehensive scenario of the forces driving TE fixation within mammalian genes is still missing.We show that intronic multispecies conserved sequences (MCSs) have been affecting TE integration frequency over time. We verify that a selective economizing pressure has been acting on TEs to decrease their frequency in highly expressed genes. After correcting for GC content, MCS density and intron size, we identified TE-enriched and TE-depleted gene categories. In addition to developmental regulators and transcription factors, TE-depleted regions encompass loci that might require subtle regulation of transcript levels or precise activation timing, such as growth factors, cytokines, hormones, and genes involved in the immune response. The latter, despite having reduced frequencies of most TE types, are significantly enriched in mammalian-wide interspersed repeats (MIRs). Analysis of orthologous genes indicated that MIR over-representation also occurs in dog and opossum immune response genes, suggesting, given the partially independent origin of MIR sequences in eutheria and metatheria, the evolutionary conservation of a specific function for MIRs located in these loci. Consistently, the core MIR sequence is over-represented in defense response genes compared to the background intronic frequency.Our data indicate that gene function, expression level, and sequence conservation influence TE insertion/fixation in mammalian introns. Moreover, we provide the first report showing that a specific TE family is evolutionarily associated with a gene function category. It is widely recognized that a large fraction of mammalian genomic DNA is accounted for by interspersed repeated elements. These sequences have been estimated to represent more than 50% of the human genome . In partOverall, TEs cover more than 45% of the human genome  but, mosOnce considered as merely junk DNA, it is now widely recognized that interspersed repeats have been playing a major role in genome structure evolution as well as having an impact on increased protein variability ,4-8 and In mammals the great majority of genes are interrupted by introns that usually outsize coding sequences by several fold. Similar to TEs, intervening regions were initially regarded as scrap DNA before being recognized as fundamental elements in the evolution of living organisms. TEs are abundant within intronic regions as well as in 5' and 3' intergenic spacers; yet, a comprehensive analysis of the forces driving TE insertion, fixation and maintenance within mammalian genes has still not been carried out. Here we show that gene features such as sequence conservation, function and expression level shape TE representation in human genes. Interestingly, we found evidence that a subset of loci involved in immune responses are enriched with MIR sequences; analysis of opossum orthologous genes, as well as of MIR frequency profiles, indicated that these TEs might serve a specific function in these loci.exp). For each gene, the TE normalized abundance (Tena) was calculated as follows:We wished to verify whether different TE types might be differentially represented depending on gene function. TE frequency varies with intron length  and GC pobs is the observed number of TEs per intron. These calculations were performed for all TE families in both human and mouse.where nTEina > 0.5 or TEna < -0.5) were classified as TE-rich or TE-poor, respectively.For each TE family, genes displaying three times more or less TE than expected (TEna (igTEna) was calculated as described for introns; for contributing gene sets the fractional igTEna deviation was then calculated as:We next used GeneMerge  to retriMean igTEna in contributing genes - mean igTEna in all genes)/|mean igTEna in all genes| GO associations are summarized in Table na (for more than one TE family). The first one is accounted for by genes involved in nucleic acid binding and transcription; these loci have, on average, high intronic MCS densities and few TEs in their flanking regions. The second functional category is represented by genes coding for cytokines/growth factors/hormones and, more generally, receptor ligands: these genes do not have, as a whole, higher than average intron conservation and, with the exception of LTR-poor genes, tend to have low igTEna. The last category (not present among Alu-poor genes) is accounted for by structural molecules, mainly represented by ribosomal proteins. These genes have extremely low MCS densities and igTEna. These same associations were retrieved for mouse genes , although no GO term was significantly associated with L1-depleted mouse genes.Data concerning significant  in humans but not in rodents.p value < 0.01) only for MIR-rich human genes  than the average . The same procedure was applied to mouse MIR-rich genes contributing to GO terms in Table p < 10-14). The difference between human and mouse in GO terms associated with MIR-rich genes, therefore, results from the cut-off we used  to define MIR-rich genes.GO terms associated with high MIR density differed between human Table  and mousWe next wished to verify whether these genes also had higher frequencies of other ancestral TEs, namely L2s and DNA transposons. The frequencies of these elements were calculated on TE-free intron size and no significant differences were identified in either human or mouse when MIR-rich genes involved in immune responses were compared to all genes (not shown); this finding suggests that relaxation of selective constraints allowing accumulation of ancestral TE insertions is not responsible for MIR over-representation in these genes. Conversely, MIR-rich ion channel introns also displayed significantly higher frequencies of both DNA transposons and L2s, indicating, therefore, that the relative enrichment in old TEs is not specific to MIRs.Canis familiaris and Monodelphis domestica (gray short-tailed opossum) annotation tables and retrieved dog/opossum genomic positions corresponding to human transcripts in our dataset. A total of 5,476 human genes could be located on the Monodelphis sequence  and, out of 85 MIR-rich immune response genes, 77 were identified in opossum (79 in dog). We then calculated the frequency of mammalian-wide MIRs within dog and opossum genes: in both species . Interestingly, in addition to mammalian-wide MIR sequences, metatherian/monotremata-specific MIR-related TEs are interspersed in the opossum genome. These latter are mainly accounted for by MON1 and MAR1 [p = 0.0023)  have been identified in the murine and human genomes . To veriWe therefore analyzed the human/mouse co-conservation profile  of human/mouse orthologous MIR instances. No significant difference was observed Figure  between na was then plotted against expression level and lowess curves calculated . To address the significance of the observed trends, 100 lowess smooths were calculated after random data permutations and empirical probability intervals were calculated . As is evident from Figure na is observed for genes above the 70th to 80th gene expression percentile. Results obtained from SAGE expression data, as well as for murine genes, gave similar results and are available in Additional data file 2.Different TE types have been reported to differentially associate with gene regions depending on expression levels . To get p < 10-16); therefore, TE density can be directly compared. Thus, for each gene we calculated the relative frequency difference as:To gain further insight, we wished to compare intronic with intergenic TE frequecies (TE number/sequence length). In fact, intergenic and intronic regions belong to the same isochore  and their lengths are correlated , as wellTEfintron/meanTEfintron) - (TEfinter/meanTEfinter).where TE distribution in mammalian genomes has been addressed in numerous studies. Yet, many questions concerning the nature of the host-element relationship still remain unanswered and a comprehensive scenario of the selective forces affecting TE fixation in mammalian genomes is still missing. In particular, genome-wide analyses of TE type distribution within and in proximity to human genes have often neglected relevant features, such as sequence conservation, gene function and expression level.vice versa [Since the precise removal of an inserted transposon is a rare event , presentce versa .HOX gene cluster is virtually deprived of TEs; the same result was obtained upon analysis of the mouse genome and interpreted in terms of TEs disturbing fine tuned regulation of developmental genes. A more recent study indicated that TE-free regions are significantly associated with genes coding for developmental regulators or transcription factors [The initial analysis of the human genome sequence  had indi factors .cis from TEs to flanking cellular sequences in plants and yeast [Our GO data indicate that functional classes associated with TE-poor genes extend well beyond highly conserved gene categories such as developmental regulators and transcription factors. In fact, some MCS-poor gene function categories also display lower than expected TEs; genes coding for structural molecules and ribosomal proteins are deprived of most TE families in both introns and intergenic spacers. These loci are mainly accounted for by housekeeping genes; if low TE representation in intronic regions might be explained by the need to reduce transcriptional costs , the reason why TEs are also excluded from intergenic spacers is more difficult to explain. One possibility is that extensive methylation of repetitive elements might exert a negative regulation on nearby gene expression with detrimental consequences for housekeeping genes. Indeed, several reports -40 have nd yeast ,42. In tnd yeast , are notnd yeast ,45), whind yeast ,47 and pnd yeast ,49) to tnd yeast ); these nd yeast . Indeed,It is interesting to notice that genes involved in immune response, which display extremely low conservation in both coding -53 and net al. [MIRs belong to a large TE superfamily referred to as CORE-SINE ; all CORet al.  suggesteet al. ,16. ThisThe last part of our work is devoted to studying the influence of gene expression level on TE distribution. In fact, despite the small population size, it has been reported that human genes show signatures consistent with selection mediated by expression levels . In partFor creation of the intron database, human genes that had been annotated in the NCBI Reference Sequence (RefSeq) collection were selected ; for mouse genes 'Provisional' entries were also included. Genomic sequences, intron/exon boundaries and intergenic regions were derived from the UCSC genome annotation database  ; a total of 238,005 and 596,018 human MCSs were retrieved in introns and intergenic sequences, respectively. In mouse, 133,458 intronic and 312,752 intergenic MCSs were identified.For the identification of human-mouse orthologous pairs, the EnsMart database  was inteMonodelphis and dog TE annotations were directly obtained from UCSC [Retrieval of opossum and dog genes and annotations was performed using UCSC tables  referrinrom UCSC  and MIR Gene associations with GO terms and their descriptions were performed by cross-referencing the UCSC hg17 kgXref table  with theMIR consensus sequences were derived from the Genetic Information Research Institute  .For calculation of the MIR relative frequency profile, human or mouse MIR instances were aligned to the consensus sequence using SWAT . MicroinFor calculation of MIR co-conservation profiles, we used the liftOver utility at the UCSC genome browser  to obtaiNlaIII site). For both human and mouse, tags were then matched to all RefSeq mRNAs and purged if they corresponded to more than one transcript.Microarray expression data for human and mouse genes were derived from previous high-throughoutput gene expression studies ,65; theySAGE libraries were obtained from the SAGE Genie website ; for botFinally, we added all counts for libraries representing the same tissue type and converted absolute tag counts to relative tag counts (counts per million).p value calculations, we performed 100 independent random data permutations of the variable on the y axis. Indeed, computing lowess smooths after random permutations of the data can be used as a reference to gauge the significance of the pattern observed on the actual data [All statistical analyses were performed using R . Locallyual data .p = 0.005 and p = 0.995 in the distribution of the 100 permutated y values considered as a Gaussian.Probability interval limits were chosen, for each x value, as corresponding to The following additional data are available with the online version of this paper. Additional data file Supplementary text presenting analysis of MCS density and TE integration frequency over evolutionary time. A supplementary figure describing the results is also provided  together with its legend.Click here for fileSupplementary Table 1 lists GO terms associated with mouse TE-poor genes. Supplementary Figure 2 shows analysis of murine MIR sequences associated with immune response genes. Supplementary Figure 3 shows gene-expression dependent variation in TE intronic abundance for human genes (SAGE data). Supplementary Figure 4 shows gene-expression dependent variation in TE intronic abundance for mouse genes (microarray data). Supplementary Figure 5 shows gene-expression dependent variation in TE intronic abundance for mouse genes (SAGE data). Supplementary Figure 6 shows intronic to intergenic relative frequency difference .Click here for file


The bioenergetic transition from glucose to ketone bodies metabolically targets brain tumors through integrated anti-inflammatory, anti-angiogenic, and pro-apoptotic mechanisms. The approach focuses more on the genomic flexibility of normal cells than on the genomic defects of tumor cells and is supported from recent studies in orthotopic mouse brain tumor models and in human pediatric astrocytoma treated with dietary energy restriction and the ketogenic diet.Malignant brain tumors are a significant health problem in children and adults and are often unmanageable. As a metabolic disorder involving the dysregulation of glycolysis and respiration, malignant brain cancer is potentially manageable through changes in metabolic environment. A radically different approach to brain cancer management is proposed that combines metabolic control analysis with the evolutionarily conserved capacity of normal cells to survive extreme shifts in physiological environment. In contrast to malignant brain tumors that are largely dependent on glycolysis for energy, normal neurons and glia readily transition to ketone bodies (β-hydroxybutyrate) for energy  The world-wide incidence of malignant brain tumors may be increasing in both children and the elderly -4. RegarMetabolic control analysis evaluates the degree of flux in metabolic pathways and can be used to analyze and treat complex diseases -16. The As abnormal energy metabolism and biological chaos are characteristics of brain tumors ,22-24, tTo manage brain cancer through metabolic targeting it is necessary to consider energy metabolism in the normal orthotopic tissue. Figure While glucose is the preferred energy substrate, neurons and glia will metabolize ketones for energy under fasting-induced reductions of blood glucose. This is a conserved physiological adaptation to prolonged food restriction and evolved to enhance survival and maintain adequate brain function while sparing proteins ,27,32-34in vitro preparations where neuronal glial interactions are disrupted and the blood brain barrier is absent /[NADH], and increasing the mitochondrial Q couple [Q]/[QH2] [2-cytochrome C reductase (site III) thus enhancing the mitochondrial proton gradient [Ketone bodies are more energetically efficient than either pyruvate or fatty acids because they are more reduced (greater hydrogen/carbon ratio) than pyruvate and do not uncouple the mitochondrial proton gradient as occurs with fatty acid metabolism . In contQ]/[QH2] ,50. The gradient . This engradient ,16. Remagradient ,50. Thesgradient .+/NADPH concentration couple is in near equilibrium with the glutathione couple, ketone body metabolism will increase the reduced form of glutathione thus facilitating destruction of hydrogen peroxide [In addition to increasing ATP production while reducing oxygen consumption, ketone body metabolism can also reduce production of damaging free radicals ,16,48. Tperoxide . The redIn contrast to normal brain that oxidizes glucose as well as ketone bodies for energy, malignant brain tumors from either humans or animal models lack metabolic flexibility and are largely dependent on glucose for energy ,23,52-582 production [In addition to glycolytic dependence, most tumors including brain tumors, express abnormalities in the number and function of their mitochondria ,66. Suchoduction ,72,73. CAkt oncogene rendering cancer cells dependent on aerobic glycolysis for continued growth and survival [Although persistent aerobic glycolysis (glycolysis in the presence of oxygen) or defects in the Pasteur effect (reduction of glycolysis in the presence of oxygen) are characteristics of many tumors, Warburg considered these phenomena too labile or too dependent on environmental conditions to be reliable indicators of tumor metabolism . Rather,survival . This issurvival . In contsurvival . It is tIn 1995, Nebeling and coworkers attempted the first nutritional metabolic therapy for human malignant brain cancer using the ketogenic diet . The ketThe patients in this landmark clinical study included two female children with nonresectable advanced grade brain tumors  . MeasuraDespite the logic of these studies and the dramatic findings, no further human studies or clinical trials have been conducted on the therapeutic efficacy of the KD for brain cancer. The reason for this is not clear but may reflect a preference of the major Brain Tumor Consortia for using "hand-me-down" drug therapies from other cancer studies rather than exploring more effective biological or non-chemotherapeutic approaches . This isThe findings of the Nebeling group were recently confirmed in a series of orthotopic mouse brain tumor models treated with the KD and dietary energy restriction ,37,81,82per se and not from the restriction of any specific dietary component such as proteins, vitamins, minerals, fats, or carbohydrates [The anti-angiogenic effects of DR arise from reduced tumor energy metabolism ,37,81,82hydrates ,88,91,92hydrates . BesidesA reduction in glycolytic energy would reduce lactate levels and hydroxyl radical production. This is important since lactate and hydroxyl radicals enhance tumor inflammation as well as cytokine production  through glial activation (microglia and astroglia) ,94. DR aBased on the differences in energy metabolism between normal brain cells and brain tumor cells, we propose a radically different approach to brain cancer management that combines metabolic control analysis with the evolutionarily conserved capacity of normal cells to survive extreme shifts in physiological environment. The adaptation to environmental extremes is conserved within the genome according to the ecological instability theory of Potts . Consequ+, K+-ATPase and Ca2+ and Mg2+ ATPases) [As a metabolic disorder involving the dysregulation of glycolysis and respiration, brain cancer is potentially manageable through abrupt changes in metabolic environment. Significant cellular energy is used to maintain the activity of transmembrane ion pumps (the NaATPases) ,97,98 (FATPases) ,97. DespATPases) . AccordiATPases) . In otheThe energy used to maintain pump function and cell viability in normal brain cells comes from either glycolysis or aerobic respiration ,99. In tThe widely held notion that brain tumor cells are somehow hardy or tough and resistant to death (programmed or nonprogrammed) may be a misconception. How can brain tumor cells, or any tumor cell for that matter, that have multiple types and kinds of genetic mutations be more fit and hardy than normal cells that possess a flexible genome and can easily transition between glycolysis and respiration for energy maintenance? The notion that tumor cells are more versatile than nontumor cells is also illogical in the context of evolutionary biology. While knowledge of tumor-associated mutations and genomic instability is of considerable academic interest, this information has produced no new or effective clinical therapies for brain tumors. Regardless of when or how genomic defects become involved in the initiation or progression of brain tumors, these defects can be exploited for the metabolic destruction of the tumor.Normal cells evolved to survive extremes in metabolic and physiological environment due largely to oscillatory changes in the physical environment . MoreoveAccording to this hypothesis, a novel strategy can be suggested for human brain tumor management that enhances the respiratory potential of normal brain cells while metabolically targeting the tumor cells. The approach would involve a sequential series of therapeutic steps and should be effective against any primary or secondary brain tumor regardless of cell of origin, anatomical location, or histological grade. Step one would lower circulating glucose levels and elevate circulating β-OHB levels through diet therapies or ketone body supplementation. Glucose ranges between 3.0–3.5 mM (55–65 mg/dl) and β-OHB ranges between 4–5 mM should be effective for tumor management in most patients. The conditions for these parameters have been described previously for children and adults and can be adjusted on a case-by-case basis ,75,103. Step two would involve surgical resection if necessary. Smaller brain tumors with reduced vascularity and clearly circumscribed boundaries should be easier to resect than larger brain tumors with poorly circumscribed boundaries and extensive vascularization . The dieFinally, step three could involve the use of either conventional or novel targeted therapies. While these therapeutic approaches might have little if any long-term benefit on malignant brain tumor management if used initially, they could be highly effective following the step one strategy after the tumor cells are weakened and metabolically isolated from the physiologically strengthened normal brain cells. Moreover, glycolysis inhibitors that would adversely affect normal cells might also be more effective following the first two steps of the proposed therapeutic strategy -109. It The objective of this new brain tumor therapeutic approach is to consistently change the physiological and metabolic environment of the tumor and the host. Only those cells with a normal flexible genome, honed through millions of years of environmental forcing and variability selection, are expected to survive extreme shifts in metabolic environment. Indeed, extreme conditions of survival and fitness will test the limits of a cell population's persistence in any given location over time . While sKD, ketogenic diet; SCOT, succinyl-CoA-acetoacetate-CoA transferase; β-OHB, β-hydroxybutyrate; TCA, tricarboxylic acid.


Normal voiding behavior in urethane-anesthetized rats includes contraction of the abdominal wall striated muscle, similar to the visceromotor response (VMR) to noxious bladder distension. Normal rat voiding requires pulsatile release of urine from a pressurized bladder. The abdominal wall contraction accompanying urine flow may provide a necessary pressure increment for normal efficient pulsatile voiding. This study aimed to evaluate the occurrence and necessity of the voiding-associated abdominal wall activity in urethane-anesthetized female ratsA free-voiding model was designed to allow assessment of abdominal wall activity during voiding resulting from physiologic bladder filling, in the absence of bladder or urethral instrumentation. Physiologic diuresis was promoted by rapid intravascular hydration. Intercontraction interval (ICI), voided volumes and EMG activity of the rectus abdominis were quantified. The contribution of abdominal wall contraction to voiding was eliminated in a second group of rats by injecting botulinum-A  into each rectus abdominis to induce local paralysis. Uroflow parameters were compared between intact free-voiding and BTX-prepared animals.Abdominal wall response is present in free voiding. BTX preparation eliminated the voiding-associated EMG activity. Average per-void volume decreased from 1.8 ml to 1.1 ml (p < 0.05), and reduced average flow from 0.17 ml/sec to 0.11 ml/sec (p < 0.05). Intercontraction interval (ICI) was not changed by BTX pretreatment.The voiding-associated abdominal wall response is a necessary component of normal voiding in urethane anesthetized female rats. As the proximal urethra may be the origin of the afferent signaling which results in the abdominal wall response, the importance of the bladder pressure increment due to this response may be in maintaining a normal duration intermittent pulsatile high frequency oscillatory (IPHFO)/flow phase and thus efficient voiding. We propose the term Voiding-associated Abdominal Response (VAR) for the physiologic voiding-associated EMG/abdominal wall response, to distinguish it from the visceromotor response (VMR) to noxious bladder distension. Electromyographic (EMG) evidence of abdominal wall contraction has been observed to accompany voiding in awake rats during natural voiding and suprapubic cystometry  as well Normal voiding in the rat has been divided into four phases . Upon thRat voiding may thus be considered as a pressurized vessel releasing its contents against an intermittently relaxing resistance. Abdominal pressurization caused by the voiding-associated abdominal wall contraction may be required in this process. We therefore hypothesized that the abdominal wall activity observed during physiologic voiding is a necessary component of normal voiding in the rat.Cystometric study either via transurethral or suprapubic catheter potentially alters the micturition reflex by direct stimulation of bladder or urethral afferents by the catheter or its placement, by supraphysiologic bladder filling rates, or by urothelial stimulation by non-urine infusion fluids. Our aim was to investigate the occurrence and necessity of voiding-associated abdominal wall activity during physiologic voiding in anesthetized rats. First, we proposed to design a free voiding model which would allow quantification of the abdominal wall EMG response and uroflow parameters during physiologic voiding in urethane-anesthetized rats with an uninstrumented lower urinary tract. Second, the necessity of the voiding-associated abdominal wall response for a normal uroflow response would be assessed by selective paralysis of the abdominal wall using botulinum toxin-A (BTX), thus removing the abdominal pressure contribution to voiding.A total of 15 urethane-anesthetized (1.2 gm/kg) female SD rats weighing 250–300 g were used for this study. Institutional Animal Care and Use Committee guidelines were observed. Physiologic data were collected, recorded, and analyzed using WinDaq software  and Microsoft Excel.All animals underwent a "free voiding" study, designed to allow quantification of the voiding response resulting from bladder filling from physiologic diuresis without instrumentation of the lower urinary tract. Vascular access for infusion and blood pressure measurement was obtained via a polyethylene catheter (PE50) placed into a femoral artery after anesthetization. EMG electrodes were placed into the right rectus abdominis, 1 cm apart longitudinally, via a small midline lower abdominal skin incision. The insulated portion of each electrode was sutured to the skin and the incision was closed. A second set of electrodes was placed into the superficial peri-urethral perineal musculature via small skin incisions, one on each side of the urethra, and secured with sutures. The animal was then placed into prone position with the urethral meatus over a 10 ml collection cup attached to a force transducer to quantify voided volume. The femoral artery catheter was connected to a Harvard PHD2000 infusion pump, with an in-line pressure transducer. The animals were left in this position until several voids had occurred. Following an initial 2 ml bolus delivered over 1 minute, animals were infused with normal saline via the arterial line at a rate of 0.08 ml/minute. This infusion rate was selected in order to produce a maximal physiologic diuresis in response to subacute volume loading [Seven of the free voiding rats were infused with normal saline via the arterial line (FV group). In eight other rats, the rectus abdominis muscle was injected with BTX (10 U), 48 hours prior to the free voiding study. Under isofluorane (2%) inhalation anesthesia, a midline skin incision was made and the skin reflected off of the underlying muscles. The toxin was injected using five evenly spaced injections into the length of each muscle belly. The skin was closed with continuous suture and the animal allowed to recover. Ampicillin 150 mg/kg intramuscular was administered prior to surgery.To confirm that the voiding-associated abdominal wall EMG response correlated with a rise in intra-abdominal pressure and that loss of this increased intensity signified a loss of voiding-associated intra-abdominal pressurization, a pressure catheter was placed intraperitoneally in one intact free voiding rat and two of the BTX group, using a 20-gauge angiocath inserted into the left abdominal flank. A fluid column was established by injecting two to three milliliters of saline, and a pressure transducer was connected to monitor intra-abdominal pressure. Position and function was checked by manually squeezing the abdomen to produce a positive pressure deflection. An example of the pressure tracing obtained in response to voiding and to abdominal squeeze is demonstrated in Figure ave) was calculated by dividing per-void volume by time for each void and taking the mean of these values. Mean values of the parameters for each animal were averaged within each group and the groups were compared using the Student t test, P < 0.05 considered significant. Mean values and standard errors of measurement (SEM) were tabulated, and results in the text are given as mean +/- SEM.Voiding and EMG parameters were analyzed for all consecutive contractions in each rat (varying from one to five) and values were averaged for each animal. EMG intensity during the VAR was assessed by integrating the EMG waveform over time. The change of EMG integral values during voiding activity was divided by the change during the same time interval pre-void, and represents the voiding-associated intensity of the rectus abdominis muscle EMG response . The useTable Figure Perineal EMG intensity level was variable among rats during voiding, with the average integrated value slightly less than unity, and did not differ between groups. While not subjected to more detailed analysis, the waveform of the perineal EMG signal changed during urine flow from a tonic discharge to bursting activity occurring at approximately 0.2 second intervals as shown in Figure This study demonstrates that intra-abdominal pressurization due to the voiding-associated abdominal wall response is a normal and necessary component of voiding in urethane-anesthetized female rats. While the voiding-associated EMG response appears to be similar to a VMR, several differences exist. The abdominal wall activation during voiding is a physiologic response, in contrast to the VMR which by definition is a response to noxious stimulus. We have observed the voiding-associated response to be associated with urine flow rather than bladder pressurization during transurethral cystometry , whereasThe increase of abdominal wall EMG intensity during voiding suggests that the voiding-associated abdominal wall contraction produces an increment of intra-abdominal pressure which contributes to the total bladder pressure available to drive urine flow. Cruz et al. demonstrated abdominal wall EMG activation in several points in the abdominal wall during voiding, including the oblique muscle groups and the rectus abdominis . Logic dThe perineal electrodes were not specifically placed into the rhabdosphincter, in order to minimize experimental impact on lower urinary tract afferent signalling. The lack of enhanced perineal muscle EMG activity during voiding may signify a lack of specific muscle activation during voiding as might be expected in the rhabdosphincter, although the change in waveform to a bursting activity with a frequency of approximately 5 Hz is consistent with IPHFO activity. This is consistent with voiding-associated perineal muscle activity observed previously . The sliBladder pressurization against intermittent urethral resistance appears to be essential for efficient voiding in the rat. Rhabdosphincter EMG activity increases during bladder filling . The tonThe bladder may be pressurized directly by detrusor contraction, or indirectly by transmitted abdominal pressure. As the bladder fills, increasing activity on pelvic afferents in response to increasing wall stress triggers efferent firing with resultant detrusor contraction, and bladder pressure begins to rise ,13. ShorIt has been demonstrated that the voiding-associated EMG activity depends upon pelvic nerve afferents . BladderThe observed responses in this study of urethane anesthetized rats should be fundamentally the same as in non-anesthetized rats. Urethane anesthesia has minimal effect on afferent and efferent functions, has little influence on neurotransmitter release, slightly depresses detrusor responsiveness, and preserves voiding reflexes at cystometry ,15-18. HVoiding-associated abdominal wall contraction is a normal and necessary component of voiding in urethane anesthetized rats. The proximal urethra may be the origin of the afferent signalling which results in the voiding-associated abdominal wall response. The importance of the bladder pressure increment due to the voiding-associated abdominal wall contraction may be in maintaining a normal duration intermittent pulsatile high frequency oscillatory (IPHFO)/flow phase and thus efficient voiding. In order to distinguish this physiologic somatic response to a normal visceral stimulus from the visceromotor response (VMR) elicited by noxious visceral stimuli, we suggest terming this aspect of voiding the Voiding-associated Abdominal Response (VAR).CPS is an Investigator for Allergan, Inc.PPS designed and conducted the experiments, and drafted the manuscript and its revisionsCPS and TBB provided guidance, oversight, and critical review of the manuscriptGTS aided in the design of the experiment and statistical analysis, provided critical oversight of the experiment, and provided critical review of the manuscript.All authors have read and approved the submitted manuscriptThe pre-publication history for this paper can be accessed here:


Horizontal gene transfer (HGT) to the plant mitochondrial genome has recently been shown to occur at a surprisingly high rate; however, little evidence has been found for HGT to the plastid genome, despite extensive sequencing. In this study, we analyzed all genes from sequenced plastid genomes to unearth any neglected cases of HGT and to obtain a measure of the overall extent of HGT to the plastid.clpP, ycf2, and rpl36 – provided strong support for taxa moving far from their organismal position. Further taxon sampling of clpP and ycf2 resulted in rejection of HGT due to long-branch attraction and a serious error in the published plastid genome sequence of Oenothera elata, respectively. A single new case, a bacterial rpl36 gene transferred into the ancestor of the cryptophyte and haptophyte plastids, appears to be a true HGT event. Interestingly, this rpl36 gene is a distantly related paralog of the rpl36 type found in other plastids and most eubacteria. Moreover, the transferred gene has physically replaced the native rpl36 gene, yet flanking genes and intergenic regions show no sign of HGT. This suggests that gene replacement somehow occurred by recombination at the very ends of rpl36, without the level and length of similarity normally expected to support recombination.Although several genes gave strongly supported conflicting trees under certain conditions, we are confident of HGT in only a single case beyond the rubisco HGT already reported. Most of the conflicts involved near neighbors connected by long branches , where phylogenetic methods are prone to mislead. However, three genes – rpl36 HGT discovered in this study is of considerable interest in terms of both molecular mechanism and phylogeny. The plastid acquisition of a bacterial rpl36 gene via HGT provides the first strong evidence for a sister-group relationship between haptophyte and cryptophyte plastids to the exclusion of heterokont and alveolate plastids. Moreover, the bacterial gene has replaced the native plastid rpl36 gene by an uncertain mechanism that appears inconsistent with existing models for the recombinational basis of gene conversion.The  Unlike the dynamic mitochondrial genome of flowering plants, which frequently incorporates plastid and nuclear sequences via intracellular gene transfer -3, the prbcL and rbcS) from a proteobacterium into the common ancestor of red algal plastids and their secondary derivatives  are a putatively monophyletic group that, with respect to plastid phylogeny, branch within the red algae, with the Cyanidiales being sister to the Porphyra/Gracilaria/chromalveolate clade  of codon-usage bias. For example, the psbB gene tree goes from 100% BP for rhodophytes being monophyletic to the exclusion of Guillardia and Odontella, using all three codon positions, to weak support for a topological change to the consensus tree when only second positions are used. First and second positions together still give strong support for the conflicting tree, indicating that first positions may also contribute a significant bias for psbB [The chromalveolates [te clade . Relatiofor psbB .psbC, atpH, psaK, dnaK, atpF, atpB, rpl31, ycf4, ycf17, ycf45, and ycf37. As above, we could not reject the conflicts outright, but we could show weakened support or induce topological changes with alternative data filtering such as using second codon positions alone or amino acids. In no case did we observe any telltale signals such as uniquely shared indels in the conflicting clades.Another well-supported conflict, psbA, is discussed in atpI, psbH, atpF, rpl16) supported the monophyly of Triticum/Saccharum/Zea, while the other four  supported monophyly of Oryza/Saccharum/Zea. In all cases there is a long branch leading to the grasses, which reflects both the lack of any close outgroups (no other monocots were included) and the well-established rapid evolution of the chloroplast genome in the stem group leading to grasses  that showed the highest level of conflict (BP = 96%), using all other monocot sequences available  for these two genes. With this improved sample, the atpI phylogeny was no longer in significant conflict with the organismal tree; instead, relationships among Triticum, Oryza, and Saccharum/Zea were entirely unresolved under an all nucleotide position ML model . Increasing taxon sampling using other available rRNA genes  gave a wThe approach used here is limited by the taxon sampling. Because our initial trees included only plastid genes, we could essentially detect HGT only from one plastid genome to another, but not transfers from other genomes into plastids. We reasoned that transfers of non-plastid genes should normally result in a long branch leading to the donee taxon within the plastid gene trees. A description of our analysis of these long-branch lineages is presented in rpl36-c gene in haptophyte and cryptophyte plastids, but not in heterokont and alveolate plastids, provides the first strong evidence for the "sisterhood" of haptophyte and cryptophyte plastids. The most parsimonious scenario is that the rpl36-c gene was transferred once to the ancestral plastid of the haptophytes and cryptophytes after this plastid lineage and the lineage(s) leading to heterokonts and alveolates diverged. Less parsimonious alternative scenarios can be imagined, but the similarity between the haptophyte and cryptophyte rpl36 genes, their position as sister lineages among the rpl36-c genes  contain only rpl36-p. Thus, a potential transfer between the Rhodopirellula lineage and the cryptophyte lineage would likely postdate the Rhodopirellula/Blastopirellula/Gemmata divergence. However, a recent HGT from an unknown donor into Rhodopirellula is also possible. On balance, based on the current bacterial sampling, the donor of the plastid rpl36-c was most likely a planctomycete related to Rhodopirellula or a proteobacterium. A cyanobacterium related to Crocosphaera watsonii is a less likely but potential donor since the Crocosphaera branches within the gamma-proteobacterial c-type group  with all rpl36-p genes in the red algal lineage, as well as with several green algae and land plants and with several rpl36-p and rpl36-c bacterial genes. A less well-conserved 6-bp 3' sequence (CAAGGT) exists in cryptophyte and some alpha-proteobacterial rpl36-c genes and in the rpl36-p gene from some cyanobacteria, land plants, green algae, and red algae. This identity extends leftwards by a further 3 bp (CGTCAAGGT) in Cryptomonas, some cyanobacteria, and Odontella. These similarities between the plastidal and bacterial rpl36-c and the rpl36-p genes in the red-plastid lineage are consistent with one or both ends being involved in recombination. A gene replacement along these lines would represent, to our knowledge, an unprecedented recombination event in terms of sequence distance. Although it is conceivable that the rpl36-c and rpl36-p genes involved in this putative recombination some 1 billion years ago shared more sequence similarity than the extant genes, rpl36-c has not diverged greatly among haptophytes and cryptophytes, and rpl36-p is quite conserved among plastids  while Rhodopirellula rpl36-c is 54% (genome is 55%). In contrast to convergence, these differences in GC content probably account for much of the divergence between the plastid and bacterial rpl36-c genes and make chance convergence much less likely.In contrast to convergent positive selection for function, GC content can have a dramatic effect on amino-acid and codon usage . Howeverrpl36-c almost certainly physically replaced rpl36-p in certain algae; but did it also functionally replace it? The two types are highly divergent from one another  of highly unusual evolutionary origin. Whereas all green plastids and those of glaucophytes contain rbcLS of expected cyanobacterial origin, red plastids possess rubisco genes of apparent proteobacterial origin. Based on phylogenetic considerations, Delwiche and Palmer [rbcLS transfers, all among eubacteria. Martin and Schnarrenberger [It was first recognized nearly 20 years ago -53 that d Palmer  argued irbcL phylogeny for a representative sampling of currently available sequences. The overall structure of this tree is very similar to that of Figure rbcL sequences have become available that show even greater similarity to red algal rbcL than those considered by that study [Nitrosospira multiformis shares 86% amino-acid identity with Gracilaria over a contiguous 381 amino-acid region of rbcL. This is within the range of identities among the red algae over this same region. The rbcL tree , perhaps 1.0–1.5 billion years ago, or in the common ancestor of cryptophytes and haptophytes (rpl36), probably not much more recently; and (iii) in both cases, the transferred genes are known  to have functionally replaced native homologs, which would explain their retention for eons as intact genes. The rpl36 transfer also serves as an important phylogenetic marker and is intriguing from a mechanistic standpoint. Thus, although HGT in plastids is extremely rare, when it happens it can be of considerable consequence and interest.Comprehensive examination of all 204 genes present in four or more of the 42 examined plastid genomes has revealed but a single new, well-supported case of HGT. This ears ago  and reviears ago -58, havid Palmer ,51-53, Vd Palmer ,51-53, Bd Palmer ,51-53, Md Palmer ,51-53, aBoth cases of plastid horizontal gene transfer occurred anciently in red algae or their secondary derivatives, while several cases of potential  or likelWe initially constructed many phylogenetic trees that gave well-supported, conflicting results suggestive of HGT, but which were ultimately deemed wrong or showed weakened support under closer scrutiny. The largest source of conflicts arose within the red algal lineage and their secondary descendants , where limited taxon sampling and early diversification of lineages led to a series of long terminal branches connected by short internal branches. This is where phylogenetic reconstruction is most prone to fail due to LBA . SubsequSome 40 cases of HGT have now been reported in plant mitochondrial genomes -15 versurpl36 and the rubisco operon are clear cases of HGT to the plastid genome. Both are ancient transfers, whereby bacterial genes have replaced native homologs and have become permanent, functional residents in their respective lineages. In contrast, the frequent (and recent) transfers in plant mitochondria occur by plant-to-plant transfer and are essentially ephemeral events, few of which seem to be of functional significance. The horizontal transfer of bacterial rpl36-c into the plastid genome represents an unprecedented example of apparent homologous recombination that defies current concepts of the sequence relatedness required to allow gene conversion/replacement to occur. The rpl36-c HGT also serves as a striking phylogenetic character that establishes an important new phylogenetic connection, linking haptophyte and cryptophyte plastids as sister groups to the exclusion of heterokont and alveolate plastids.This study confirms and quantifies the hypothesis that HGT is rare in plastids. Only Eimeria tenella (AY217738), Euglena gracilis (X70810), Euglena longa (AJ294725), Guillardia theta (AF041468), Toxoplasma gondii (U87145), Cyanophora paradoxa (U30821), Cyanidioschyzon merolae (AB002583), Cyanidium caldarium (AF022186), Gracilaria tenuistipitata (AY673996), Porphyra purpurea (U38804), Odontella sinensis (Z67753), Adiantum capillus (AY178864), Amborella trichopoda (AJ506156), Anthoceros formosae (AB086179), Arabidopsis thaliana (AP000423), Atropa belladonna (AJ316582), Calycanthus floridus (AJ428413), Chaetosphaeridium globosum (AF494278), Chlamydomonas reinhardtii (BK000554), Chlorella vulgaris (AB001684), Epifagus virginiana (M81884), Lotus japonicus (AP002983), Marchantia polymorpha (X04465), Medicago truncatula (AC093544), Mesostigma viride (AF166114), Nephroselmis olivacea (AF137379), Nicotiana tabacum (Z00044), Nymphaea alba (AJ627251), Oenothera elata (AJ271079), Oryza sativa (X15901), Physcomitrella patens (AP005672), Pinus koraiensis (AY228468), Pinus thunbergii (D17510), Psilotum nudum (AP004638), Saccharum officinarum (AP006714), Spinacia oleracea (AJ400848), Triticum aestivum (AB042240), Zea mays (X86563), Plasmodium falciparum , Panax ginseng (AY582139). In addition, the sequence of the Pisum sativum plastid genome was provided by John C. Gray (unpublished data), and the Vitis vinifera coding sequences were extracted and pieced together (unpublished result) from all NCBI nucleotide databases including dbEST [Pisum sativum, Vitis vinifera, and Medicago truncatula.EMBL-Bank files for the following 40 plastid genomes were retrieved from the European Bioinformatics Institute: us AY1788, AmborelIn total, 5676 protein, tRNA and rRNA genes were extracted from the 42 plastid genome sequences. A BLAST  databaseProtein and nucleotide alignments were made for each of the gene families using MUSCLE software  with unlML models for each gene family were determined using the likelihood-ratio test criterion of MODELTEST  except wA consensus plastid tree  was usedrpl36 (DQ365944–DQ365946) and ycf2 (DQ370441–DQ370447) were obtained using standard PCR and sequencing protocols [rpl36 isolation) were generously provided by Chris Lane and John Archibald. Angiosperm DNAs (for ycf2 isolation) were isolated [Several new sequences for rotocols . Cryptoprpl36 and ycf2 sequence data, and drafted the manuscript (text and figures). DWR and JDP jointly directed the project, and JDP contributed substantially to the final manuscript. Both authors read and approved the final manuscript.DWR carried out all the analyses, wrote the new software used in this study, generated the new Cyanophora, green algae, and red algae; among bryophytes; among chromalveolates; and whether Amborella plus Nymphaea or Amborella alone is the sister of all other angiosperms).Consensus plastid phylogeny. Shown is the plastid phylogeny used in this study, based on the current literature. Dashed and solid vertical brackets denote paraphyletic and monophyletic groups, respectively. Although the tree is shown as entirely resolved, some parts are not well supported  This shows that secY and rps13 from plastids which contain the c-type rpl36 are more similar to secY and rps13 genes from red algae and cyanobacteria than to potential rpl36 donors, Rhodopirellula and proteobacteria. Amino acids that conflict with the consensus amino acid at each position are colored according to the key. The similarity between the c-type rpl36 genes in the haptophyte and cryptophyte plastids and those from Rhodopirellula and proteobacteria is also apparent. Note that rpl36 is flanked by secY and rps13 in plastids of red algal origin, but the c-type rpl36 in bacteria is not flanked by these genes. (B) Amino-acid alignment of c-type and p-type rpl36 genes. Note the three apicomplexans included in the alignment . At the bottom is shown which residues make contact with the 23S rRNA in the ribosome crystal structure of Escherichia coli, which has a p-type rpl36.Amino-acid alignment of Click here for filerpl36 tree obtained in the initial phylogenetic screen. This shows the Guillardia rpl36 gene going arbitrarily with Pisum with weak support given the poor taxon sampling and low information content of this gene. See Figure The poorly resolved Click here for fileOenothera elata ycf2 gene error in the genome sequence. Discussion of the Oenothera elata ycf2 gene error in the genome sequence.Discussion of the Click here for filepsbA conflict in the red algal lineage. Results of the psbA conflict in the red algal lineage.Results of the Click here for fileatpI gene. (A) The topology and support values with the original taxon sampling. The conflicting node is indicated by the bold 96; (B) after more monocot taxa are added Triticum and Zea move apart.Resolution of conflicts within grasses by adding taxa for the Click here for fileList of gene trees having branches ≥ 4 SD above the mean branch length for a given tree and text describing analysis of long branch taxa. The text describes the analysis of long-branch taxa. The table gives the number of standard deviations above the mean, the corresponding gene and the group that the long-branch leads to.Click here for fileDiscussion of chromalveolate hypothesis and algal phylogeny. Discussion of chromalveolate hypothesis and algal phylogeny.Click here for fileEmiliania based on all plastid genes.Multigene chromist plastid phylogeny. Results of the phylogenetic placement analysis of the haptophyte Click here for file(A) The topology found using MrBayes with 21,659 plastid amino-acid positions shared among the chromophytes, Gracilaria, and Porphyra. Invariant sites and gamma distributed rates were used with the Cprev model [(B) The topology was found as in (A) except that Guillardia and Emiliania were constrained to be monophyletic. The node corresponding to the chromist clade had a posterior probability of 0.98 and the other nodes had 1.0.Plastid genes supporting the sisterhood of either haptophytes and heterokonts or haptophytes and cryptophytes. The histograms list the genes favoring the given topology over the other according to the codeml amino-acid ML score with the WAG matrix  and gammev model . The posClick here for file


Genetic recombination is a fundamental evolutionary mechanism promoting biological adaptation. Using engineered recombinants of the small single-stranded DNA plant virus, Maize streak virus (MSV), we experimentally demonstrate that fragments of genetic material only function optimally if they reside within genomes similar to those in which they evolved. The degree of similarity necessary for optimal functionality is correlated with the complexity of intragenomic interaction networks within which genome fragments must function. There is a striking correlation between our experimental results and the types of MSV recombinants that are detectable in nature, indicating that obligatory maintenance of intragenome interaction networks strongly constrains the evolutionary value of recombination for this virus and probably for genomes in general. Genetic exchange between organisms, called recombination, occurs in all biological kingdoms and is also common in viruses in which it may threaten the long-term control of important human pathogens such as HIV and influenza. Although recombination can produce advantageous gene combinations, bioinformatic analyses of bacterial genomes have suggested that recombination is not well tolerated when it involves exchanges of genes that interact with a lot of other genes. Using laboratory-constructed recombinants of a small plant virus called MSV, Martin and co-workers provide the first direct experimental evidence that the evolutionary value of exchanging a genome fragment is constrained by the number of ways in which the fragment interacts with the rest of the genome. They note that fitness losses suffered by artificial MSV recombinants increase with decreasing parental relatedness. Furthermore, these losses accurately anticipate the patterns of genetic exchange detectable in natural MSV recombinants, suggesting that they accurately reflect the impact of deleterious selection on natural isolates of the virus. Genetic recombination may predate the evolution of cellular life  and is tMastrevirus) as a model organism to investigate the effect of genomic recombination on viral fitness.Here we provide experimental support for these hypotheses using the small single stranded plant DNA virus, Maize streak virus  from four pairs of MSV isolates sharing genome-wide nucleotide sequence identities of 98%, 95%, 89%, and 78% .–22.13–22 CP gene .MP, CP, LIR, and Rep are 15.3%, 8.3%, 2.7%, 3.3%, and 3.8% respectively. To avoid any biases due to “cherry picking” the Ti values we used to compare the different genome regions, we examined estimated recombination tolerances over the range of Ti values between 0.99 and 0.7. There is good correlation between the number of known MSV intergenome component protein–protein and protein–DNA interactions for different genome regions  and recombination tolerance estimated for these same regions over the entire Ti range between 0.94 and 0.7 .To determine whether the known network of interactions occurring during an MSV infection is anticipated by our Ti data, it was necessary to first extract a relative modularity score for each of the MSV genome regions from the Ti plots. To do this we fitted quadratic equations to the plots see  to estimMastrevirus sequences in GenBank for evidence of recombination. In each of the five genomic regions , we identified evidence for unique recombination events involving MSV isolates or the closely related African streak viruses. We only retained evidence of recombination events detectable in genomes with proven viability (as determined by the existence of infectious clones of these genomes). For each recombination event involving two identifiable parental sequences and comprising two easily identifiable breakpoints both unambiguously within one of the five defined genomic regions, we used the identified parental sequences to infer the number of nucleotide differences between the transferred sequence and the sequence it replaced  was significantly correlated with equivalent sets of values derived from the quadratic regressions shown in R2 > 0.96, p < 0.01, Spearman's rho = 0.9, p = 0.037 over the Ti range 0.95–0.7).To investigate whether experimentally determined Ti values provide any insights into real processes that influence the survival of recombinants in nature, we examined all available replaced . The greBy demonstrating a negative correlation between the relative modularity of defined genomic regions and the complexity of interactions in which they are involved we have provided experimental support for the complexity hypothesis . This hyThe progressive decrease in tolerance of recombination with increasing divergence of exchanged sequences observed in The successful inheritance of genomic fragments through recombination is expected to depend on the maintenance of important intragenome interactions. After all, the exchange of a genome fragment could be seen as a simultaneous introduction of multiple mutations. Negative or purifying selection should remove those recombinants that break the epistatic interactions that define the architecture of a particular genome, whereas genetic drift might permit the survival and spread of “neutral” recombinants. Alternatively, positive selection should favour the spread of rare recombinants with improved genomic interactions. The genomic interactions in the  parental viruses used in these experiments have most likely been optimised through selection over long evolutionary periods. None of the recombinants generated from these viruses was more fit than the fitter of its parents, which is expected if negative selection is the dominant force that now maintains the integrity of these genomes.The relative degree of modularity that we demonstrated experimentally for each genome component appears to be reflected in the recombination events detected within the same regions in natural viruses represented in GenBank. This correlation is surprising because the natural recombinants—unlike the recombinants we constructed in the laboratory—involve exchanges of fragments of genes or intergenic regions. Such exchanges may disrupt intraprotein or intra-intergenic region interactions as well as interaction networks amongst whole genes and intergenic regions. Survival of the natural viruses with detectable recombination events in coding regions presumably depended on their inheritance of sequences that did not overly disrupt either intraprotein or intergene/intergenic region interaction networks; survival of natural intergenic region recombinants and those we generated in the laboratory would have been subject only to the latter constraint. The correlation between our experimental results and the inferred natural recombinants may indicate that maintenance of intergenome component interactions is the principal determinant of recombination tolerance (at least for MSV and closely related viruses). Alternatively, a requirement for the preservation of both intergenome component, and intragene, interaction networks has a net effect that is difficult to distinguish from either constraint operating alone.We have provided experimental support for the complexity hypothesis by demonstrating a relationship between the relative modularity of defined genomic regions and the complexity of interactions in which they are involved. The striking correlation between our experimental results and the types of recombination observed in nature lends credence to the notion that these detectable modularity differences are evolutionarily relevant. Our results also suggest that the degree of similarity between an inherited sequence and the sequence it replaces is an important additional determinant of recombinant fitness. Whereas recombination can substantially increase the evolutionary options of an organism, the obligatory maintenance of co-evolved interaction networks may severely restrict its evolutionary value.CP gene start codons of the MSV isolates MSV-Set and MSV-Kom to obtain SNco and KNco, respectively. Reciprocal MP gene recombinants were obtained by exchanging BamHI-NcoI restriction fragments containing the entire MP between MSV-SNco and MSV-KNco. Reciprocal CP recombinants were obtained by exchanging NcoI-NcoI restriction fragments containing the entire CP between MSV-SNco and MSV-KNco. Agroinfectious partially dimeric clones of recombinant viruses were constructed as previously described [We have previously described the construction and symptom analysis of 15 reciprocal recombinant pairs for the MSV isolates MSV-Mat, MSV-Kom, MSV-R2, and MSV-VW . PCR mutescribed .The fitness of recombinant and parental viruses as indicated by ICLA was determined in maize by agroinoculation of 3-d-old cv. Jubilee seedlings with image analysis quantification of ensuing disease symptoms . An ICLAMastrevirus sequences were obtained from GenBank and aligned using POA [p-value cutoff of 0.05, and a requirement that any potential event be detectable by two or more methods.All available sing POA  with gapsing POA , Geneconsing POA , RecScansing POA , Maximumsing POA , Chimaersing POA , and Sissing POA  methods sing POA . The anahttp://www.ncbi.nlm.nih.gov/Genbank) accession numbers for the genome sequences are CSMV (NC001466), DSV (NC001478), MiSV (NC003379), MSV-Ama (AF329878), MSV-Gat (AF329879), MSV-Jam (AF329887), MSV-K (X01089), MSV-KA (AF329885), MSV-Km (AF395891), MSV-Kom (AF003952), MSV-MakD (AF329884), MSV-Nig (X01633), MSV-Pat (AF329888), MSV-Raw (AF329889), MSV-Reu (X94330), MSV-SA (NC001346), MSV-Sag (AF329880), MSV-Set (AF007881), MSV-Tas (AF239962), MSV-VM (AF239961), MSV-VW (AF239960), PanSV-Kar (NC001647), PanSV-Ken (X60168), SSEV-Ben (AF039529), SSRV (NC004755), and SSV-N (NC003744).The GenBank (


There is increasing evidence that programmed cell death can be triggered during cardiopulmonary bypass (CPB) and may be involved in postoperative complications. The purpose of this study was to investigate whether apoptosis occurs during aortic valve surgery and whether modifying temperature during CPB has any influence on cardiomyocyte apoptotic death rate.20 patients undergoing elective aortic valve replacement for aortic stenosis were randomly assigned to either moderate hypothermic  or mild hypothermic  CPB. Myocardial samples were obtained from the right atrium before and after weaning from CPB. Specimens were examined for apoptosis by flow cytometry analysis of annexin V-propidium iodide (PI) and Fas death receptor staining.In the ModHT group, non apoptotic non necrotic cells  decreased after CPB, while early apoptotic  and late apoptotic or necrotic (PI positive) cells increased. In contrast, no change in the different cell populations was observed over time in the MiHT group. Fas expression rose after reperfusion in the ModHT group but not in MiHT patients, in which there was even a trend for a lower Fas staining after CPB (p = 0.08). In ModHT patients, a prolonged ischemic time tended to induce a higher increase of Fas (p = 0.061).Our data suggest that apoptosis signal cascade is activated at early stages during aortic valve replacement under ModHT CPB. This apoptosis induction can effectively be attenuated by a more normothermic procedure. Cardiomyocyte apoptosis has been involved in the pathophysiology of various cardiovascular diseases such as ischemic cardiomyopathy, hibernating myocardium, heart failure, reperfusion injury, and transplant rejection ,2. Recenin situ terminal deoxynucleotidyl-transferase mediated dUTP nick end-labeling), has a poor positive predicted value, as it labels not only fragmented DNA but also DNA in the process of repair as well as necrotic tissue [One of the reasons for scarce data concerning apoptosis and its pathophysiological consequences during cardiac surgery is the inferiority of the various techniques used to detect apoptotic cell changes. The most commonly used method, histochemical staining of the fragmented DNA by the TUNEL assay  blood cardioplegia . Patients were randomly assigned to one of two groups: moderate hypothermic CPB  or mild hypothermic CPB . After institution of CPB, patients in the ModHT group were cooled until core  temperature reached 28°C, whereas the temperature of patients in the MiHT group was just let to drop until it leveled off spontaneously. Baseline biometric characteristics and CPB data of trial groups are shown in Table 3) were obtained from the right atrial appendage of each patient before the start of CPB and after weaning from extracorporeal circulation. To minimize technical influences on apoptotic and necrotic measurements, particular care was taken not to mechanically irritate the site of atrial tissue harvesting with the forceps and with the venous cannula. Blood samples were also taken from the coronary sinus at the same time of heart biopsy collection for analysis of necrosis.Using a sharp scalpel, myocardial biopsy specimens . Cells were washed with cold phosphate buffer saline (PBS) to exclude cell debris and resuspended in culture medium . This procedure rendered a preserved viability cell population as judged by trypan blue dye exclusion. To be sure that cardiac cells were specifically quantified, intracellular expression of the specific protein -connexin 43- was analyzed. After fixation and permeabilization with IntraStain kit , cells were incubated with connexin 43 rabbit polyclonal antibody . Binding was revealed by phycoerythrin conjugated goat anti-rabbit IgG  and flow cytometry. The antibodies were initially titrated for optimal concentration, and cells incubation with only phycoerythrin-antibody was used as negative control.Apoptosis was measured by staining cells with a combination of fluoresceinated (FITC) annexin V and propidium iodide (PI) and by Fas death receptor quantification on cell membrane.2+-dependent phospholipid-binding protein that has a high affinity for phosphatidylserine. Because externalization of phosphatidylserine occurs in the early stages of apoptosis, annexin V staining can identify apoptotic cells earlier than assays based on nuclear changes like DNA fragmentation, and precedes the loss of membrane integrity which accompanies the latest stages of cell death resulting from either apoptotic or necrotic processes. Therefore, staining with annexin V in conjunction with vital dye such as PI allows to identify early apoptotic cells  and non apoptotic non necrotic cells  . Next, 5 μl of annexin V-FITC  were added and incubated for 15 minutes at room temperature preserved of the light. Then, 100 μl of PI-PBS solution (50 mg/ml)  were added and immediately data acquisition was made in the flow cytometer in order to avoid cell damage and the diffusion of PI through cell membrane.Changes in the plasma membrane are one of the earliest morphological features of apoptosis. In apoptotic cells, the membrane phospholipid phosphatidylserine is translocated from the inner to the outer leaflet of the plasma membrane, thereby exposing phosphatidylserine to the external cellular environment. Annexin V is a 35–36 kD Ca) Figure . The assFas molecule APO-1/CD95) belongs to the tumor necrosis factor receptor superfamily and may induce apoptosis by Fas-Fas ligand (Fas-L) binding. The Fas/Fas-L system is an essential part of the death receptor pathway regulating the induction of a rapid apoptosis independent of mitochondria. We measured Fas by membrane cell staining with FITC conjugated anti-CD95 monoclonal antibody (BD Biosciences) .Data are presented as mean ± standard deviation. The χGroups were well matched in terms of age, sex, prevalence of cardiovascular risk factors and atrial fibrillation, preoperative left ventricular function, and duration of CPB and aortic crossclamping Table .Changes in cardiomyocyte death status during the operative procedure are summarized in Table Apoptosis has been reported not only to occur during open heart surgery but also to be related to postoperative myocardial stunning, renal insufficiency, and cerebral dysfunction ,4,9. CarStrategies towards a less apoptogenic procedure appear to be a goal to achieve excellence in cardiac surgery and may include shortening of ischemic and CPB times, increased use of intraaortic balloon pump and noncatecholamine inotropes, addition of antiapoptotic medication, or modulation of intraoperative temperature.bak) and promoting antiapoptotic factors (Bcl-x) [Hypothermia improves resistance to ischemia-reperfusion injury in the myocardium. Protection afforded by low temperature may be linked to a wide variety of effects, including reduction of ATP store depletion, inhibition of endothelial cell expression of E-selectin, modification of cell death signaling pathways, and preservation of gene expression for mitochondrial proteins . Neverth (Bcl-x) . This fi (Bcl-x) -22.per se apoptogenic state of CPB, and that might be involved in the decreased systolic performance often observed with cooling [Our results suggest an additive pro-apoptotic effect of moderate hypothermia that may superimpose on the  cooling ,18. Two 2+ [The annexin V staining as a method for cell death analysis has been mainly applied in lymphoid disorders, but there is increasing evidence that it is also useful for the detection of apoptotic cardiomyocytes ,23,24. WThe biological relevance of Fas has been mainly proved in relation to T lymphocytes activity in the context of autoimmune and hematopoietic diseases ,8. NeverIn conclusion, our data show that apoptosis signal cascade is activated at early stages in human myocardium during aortic valve replacement under ModHT CPB. This apoptosis induction can effectively be attenuated by a near-normothermic strategy. In our experience, the biochemical benefit of mild hypothermia is not apparently based on the reduction of bypass times or the use of less postoperative inotropic support, and should response to a direct impact of temperature on cell membrane stability and death receptor signaling pathway.Heart tissue samples were harvested from the right atrial appendage with the intention of obtaining an appropriate amount of tissue without impairing cardiac function. This strategy has been previously reported for the study of apoptosis during CPB . NeverthThe percentage of cardiomyocytes that have initiated the apoptotic program at the time of first sample acquisition (preCPB) seems remarkably high in both groups , which may indicate a lack of specificity of both techniques. Although tissue damage during biopsy collection might have contributed to artifact the results, we hypothesize that our data may reliably reflect the state of apoptosis in atrial myocardium just before CPB. In fact, contributing factors such us anesthesia induction, sternotomy, or cannulation may trigger the apoptotic cascade before the start of CPB, as it has been suggested by previous works . Besides


We performed an experimental study on slow releasing anticancer drug implantation treatment as a new therapy for hepatocellular carcinoma. Hydroxyapatite (HAP) was chosen for the carrier material and doxorubicin hydrochloride (DOX) for anticancer agent. DOX-HAP was produced by adsorbing DOX to porous HAP particles of 1375 +/- 125 microns diameter using the freeze drying method. In vitro experiments showed slow release of the drug resulting in the steady release of DOX from HAP for 1 month duration. In healthy white rabbits with DOX-HAP implantation in the liver, serum DOX was not detectable, and DOX release rate was stable at the implanted region after 7, 14, and 21 days. When DOX-HAP  was administered to mice with sarcoma 180, an improved survival rate was observed without acute toxicity. We also found that VX2 liver tumour growth on white rabbit was inhibited by implantation of DOX-HAP, without acute toxicity. We hope that DOX-HAP implantation therapy will open up new avenues for the treatment of hepatoma.