Off-pump coronary artery bypass grafting has been accused of possibly compromising graft patency. Sixteen slice computed tomography has shown good diagnostic accuracy in the assessment of coronary bypass graft patency when compared with conventional coronary artery angiography and is less invasive. The study hypothesis is that coronary artery bypass grafting (CABG) performed without cardiopulmonary bypass (Off-Pump) has equivalent early graft patency as if performed with cardiopulmonary bypass (On-Pump) and may have reduced complication rate.Prospective Randomized Comparison of Off-Pump and On-Pump MultI-vessel Coronary Artery BypasS Surgery (PROMISS) is a controlled, single blinded, single centre clinical trial, comparing early graft patency using 16-slice computed tomography in patients with multi-vessel coronary artery disease operated either without or with extracorporeal circulation. Inclusion criteria are multivessel disease with an indication for first time, isolated, non emergent coronary artery bypass grafting with a minimum of three distal anastomoses. Secondary end points are peri-operative mortality, combined morbidity, length of stay, neuro-cognitive testing at 6 weeks and adverse events, stress test and quality of life at 6 months and one year. The sample size of one hundred and fifty patients was calculated in order to enable the detection of a 5% difference in graft patency, with 80% power, considering a minimum of 3 distal anastomoses per patient. Enrolment started in April 2005 and ended July 2007 with study closure in July 2008.The The PROMISS trial aims to shed new light on the effect of Off-Pump as compared to On-Pump coronary artery bypass surgery on graft patency, assessed by multidetector computed tomography, in unselected patients with multivessel coronary artery disease.Current Controlled Trials ISRCTN58800729 Coronary artery bypass surgery is a proven and effective mode of treatment for ischaemic heart disease. It provides relief of symptoms and increases survival, in particular in patients with left main disease and two or three vessel disease including the proximal left anterior descending artery .Coronary artery bypass grafting (CABG) can be performed in several ways but in the majority of cases it is still performed with the assistance of cardiopulmonary bypass (CPB), aortic cross clamping and cardioplegia induced cardiac arrest . Coronary artery bypass grating can also be performed without CPB ("off-pump" or OPCAB) and this method's application varies from around 20% in the USA and Europe to more than 50% in India and Japan.As a consequence of increased life expectancy and a more widespread use of coronary angiography with exponential growth of percutaneous coronary interventions in ischaemic heart disease, patients proposed for CABG are older and suffer from more co-morbid conditions. In spite of this, continuous incremental improvements in peri-operative patient management, anaesthetic and surgical techniques have led to better results . HoweverMost studies show that OPCAB is associated with less cardiac, pulmonary, renal or neurologic complications and reduces blood transfusion and hospital stay compared with "on-pump" coronary bypass surgery -7. HowevIn general, there has been difficulty in accumulating scientific evidence for OPCAB benefit, due to low incidence of events , therefore requiring very large samples or high risk populations to demonstrate a statistically significant improvement .On the other hand, "off-pump" CABG has been accused of compromising completeness of revascularisation and graft patency by its increased technical difficulty, particularly in grafting the left ventricular lateral wall . Indeed,Coronary artery bypass grafting has an important role in the treatment of coronary artery disease as shown by series with long term results . HoweverProspective Randomized Comparison of Off-Pump and On-Pump MultI-vessel Coronary Artery BypasS Surgery (PROMISS) derives from current discordant data in the literature regarding the potential inferior graft patency when CABG is performed off-pump as compared with the arrested heart on-pump procedure [The justification for therocedure -15. Therrocedure -15.Many procedural and surgeon related factors  can account for these differences so it is important to evaluate both off and on-pump coronary bypass surgery using standardised techniques, unbiased assessment of results and an accurate patency control method.Until recently, coronary angiography was the only method to assess coronary artery bypass graft patency. This technique is stressful, causes discomfort and has local , neurological  and cardiac  complications in up to 1% of cases . From anRecent advances in multidetector computed tomography (MDCT) technology, namely with reduced acquisition time and improved spatial resolution, has permitted its application to the detection of coronary artery disease and bypass graft evaluation . Relativprimary objective is to compare early coronary bypass graft patency by 16 slice computed tomography in patients with multivessel coronary artery disease undergoing first time coronary artery bypass grafting with (on-pump) and without (off-pump) cardiopulmonary bypass.PROMISS Secondary endpoints are:1- Mortality and morbidity in patients operated with on-pump and off-pump.2- Comparative changes in neuro-cognitive performance between base line and 4–6 weeks by a battery of neuropsychological tests.3- Evaluation of functional status, ischaemic threshold by stress test, quality of life and adverse events at 6 months and one year.Finally, as tertiary end point we aim to analyse hospital costs.PROMISS is a prospective, randomized, single blinded, single centre study.Patient inclusion, surgery, post operative care, multidetector computed tomography and follow-up are conducted at Hospital da Cruz Vermelha Portuguesa. The principal investigator designed the study and is the surgeon responsible for performing all on-pump and off-pump operations. The principal investigator has a large experience in on-pump CABG and has performed more than 300 OPCAB surgeries before initiating the study.PROMISS is registered in The International Society for Clinical Randomized Trials (ISRCTN58800729), it is conducted in accordance with the principles of The Declaration of Helsinki, with the Portuguese laws and rules and subscribes to the principles outlined in the International Conference on Harmonisation of Good Clinical Practice .All patients receive full explanation of study objectives, the operations to be performed, with or without CPB according to randomization, its risks and benefits and signed the informed consent form.Any death during the study period requires the hospital ethical commission to be informed.All patients that comply with the inclusion and exclusion criteria are considered for enrolment in PROMISS Table . InclusiNo patient is excluded because of recent myocardial infarction, ventricular dysfunction, or poor quality target vessels . No patient is excluded for associated morbid conditions with the exception of renal insufficiency (because of iodine administration) and atrial fibrillation (multidetector computed tomography imperative).During the study period, a patient diagram flow is used indicating patients screened, patients enrolled and reasons for exclusion, patients randomized and patients available for MDCT and follow up according to the CONSORT recommendations  .The informed consent is signed before randomization. Randomization is performed by the method of random permuted blocks. A sealed envelope containing the patient's number is opened in the operating room before the beginning of the operation.Cross-over from one group to another is possible in case patient safety is deemed at risk, a condition that is estimated to occur in less than 5% of the cases:• From on-pump to off-pump group in case of calcified ascending aorta.• From the off-pump to the on-pump group in case of refractory hemodynamic deterioration.Patients who cross over from off-pump to on-pump are analysed in the original off-pump group for which he or she was randomized in intent to treat analysis.Premature exit from the study is possible at any moment at patient's will or because of inclusion/exclusion criteria violation.Premedication, anaesthetic protocol, patient opening and closing, harvesting and graft anastomoses techniques are the same in both groups.On-pump CABG uses a conventional roller pump cardiopulmonary bypass machine with an arterial filter, continuous ultra filtration, and a central temperature that is allowed to drift to 34°C. Myocardial protection is provided by 34°C antegrade blood-potassium cardioplegia delivered every 25 minutes.Off-pump coronary artery bypass grafting uses a deep pericardial stitch and vacuum stabilizers for exposure. Intra coronary shunts are systematically used.The planned/performed distal anastomoses index and the reason for eventual incomplete revascularisation as well as target coronary artery quality  are noted in the two groups.Post operative care is under the responsibility of the same intensive care team and follows the rules and protocols of the hospital ICU.Treatment group is blinded to patients, family and investigators responsible for the MDCT angiographic control, neuro-psychological tests and follow-up.Demographic data, pre operative, intra operative, post operative and follow up variables are reported in a dedicated Case Report Form. Event adjudication is made by hospital physicians according to standardized definitions Table . ProtocoThe study main end point, comparison of graft patency at 4 to 6 weeks between the two groups, is performed by a GE Lightspeed 16 slice multidetector computed tomography using a standard protocol already described . A cardiEach graft is independently graded using a three grade scale: occluded graft, graft stenosis of 50% or more, and stenosis < 50% or no stenosis at proximal anastomoses (if any), body graft and distal anastomosis .Graft occlusion is defined as absence of contrast material along the course of the graft, through the graft anastomosis to the native coronary artery or to the following graft segment and native vessel. In sequential bypass grafts, each anastomosis of one graft is counted as a separate graft. The overall graft score is the worst of the three subcsores. Graft patency is evaluated according to graft material, site, as well as number of anastomoses patent per patient.Secondary end points are:1- Revascularisation index: number of planned distal anastomoses/number of performed distal anastomoses.2- Adverse events at hospital discharge and at 4–6 weeks • Digit Symbol • Grooved pegboard test• Auditory verbal learning test• Complex Figure Test• Trail making test A and B• Letter Word Fluency• Controlled Oral Word Association (COWA)• Hospital Anxiety Depression Scale (HADS)• Beck Depression InventoryThe assessment of 50 healthy demographically matched control subjects, with the same test battery and test-re-test procedures, will enable the calculation of T -scores for each neuropsychological evaluation. Decline will be defined by a drop equal or greater to 15 points.4- Follow up at 6 months and 1 year will compare functional status, stress test, adverse events and quality of life by EuroQOL between the two groups.is the analysis of costs: direct patient care related in hospital costs.PROMISS was planned as a non inferiority study of one intervention (OPCAB) relative to the reference operation (on-pump). This study was designed with adequate power  to detect an absolute difference in patency rates between groups in either direction as small as 5%. The calculation resulted in a total of 426 units . Since each patient has by the inclusion criteria at least 3 grafts, 71 patients in each group are necessary (213 grafts) for a total of 142 patients. Considering the possibility of 4 patients' withdrawal in each group, a total sample of 150 patients was established.2.Discrete data are presented as numbers and percentages; continuous data are presented as median, mean (SD) and compared using t test or the Mann-Whitney test. Dichotomous morbidity and mortality outcomes are analyzed using the Fisher exact test. Ordered categorical outcomes are compared between groups using the Mantel Haenszel χDichotomous patency outcomes or graft stenosis > 50% are analyzed using the Fisher exact test. Mean number of patent anstomosis per patient is compared using the t test or the Mann-Whitney test.Prospective Randomized Comparison of Off-Pump and On-Pump MultI-vessel Coronary Artery BypasS Surgery (PROMISS) was designed to try to answer the question of whether or not coronary artery bypass grafting performed off-pump has the same early graft patency as if performed on-pump in arrested hearts.The The sample size was calculated in order to detect a +/- 5% difference between the two techniques. This trial includes only patients with multi vessel disease with an indication to perform at least 3 distal coronary artery anastomoses for two reasons. Firstly to compare the two techniques in terms of completeness of revascularisation since various series have reported a lower number of grafts/patient in OPCAB relative to conventional on-pump surgery . SecondlFew studies have compared OPCAB versus CABG with CPB among randomly assigned patients unselected for coronary anatomy, ventricular function or co-morbidities except renal insufficiency. All surgeries using standardized techniques were performed by a single surgeon experienced with both methods in an attempt to eliminate technical variability.This is the first randomized study comparing early graft patency in off-pump and on-pump coronary bypass surgery using 16 slice multidetector computed tomography in patients with multivessel disease. Although 64 slice multidetector computed tomography has improved temporal and spatial resolution, there is ample evidence that, with appropriate heart rate reduction, 16 slice MDCT is able to accurately detect graft occlusion and stenosis compared to conventional coronary angiography ,28,29.Limitations of this study include the fact that physicians involved in patient care, except those assessing graft patency, are not blinded to treatment method, which can eventually bias treatment decision making and length of stay. Another limitation is the single surgeon type of study which may prevent generalizability of the results. Finally, the assumption made for sample size calculation that graft patency within a patient is an independent research unit may not always apply requiring application of statistical adjustment.The Prospective Randomized Study of graft patency in Off-pump and On-pump MultI-Vessel coronary artery bypasS Surgery (PROMISS) using multidetector computed tomography, has the potential to shed new light on the ongoing debate regarding these two techniques and therefore may have implications on the revascularisation strategy.Prospective Randomized Study of graft patency in Off-pump and On-pump MultI-Vessel coronary artery bypasS Surgery; TIA: Transient Ischemic Attack; ULN: Upper Limit of Normal of Laboratory value.ARDS: Acute Respiratory Distress Syndrome; CABG: coronary artery bypass grafting; CAD: Coronary Artery Disease; ICU: Intensive Care Unit; MDCT: Multidetector Computed Tomography; OPCAB: Off-Pump Coronary Artery Bypass; PROMISS: The authors declare that they have no competing interests.All authors read and approved the final manuscript. Specifically, MSU principal investigator, conceived and designed the study and drafted the manuscript. FM is responsible for performing computed tomography studies and helped draft the manuscript. SC conceived and coordinated the neurocognitive evaluation and helped with statistical analysis. MPM helped draft the manuscript and supervised the research project.


Sir,et al :615–616). However, the contrary may also occur when an orally 5-FU derivative is combined with folinic acid. Recently, we observed an interesting case on decreased phenytoin levels associated with concurrent oral tegafur/uracil/calcium folinate therapy. A 53-year-old Caucasian male with a history of rectal carcinoma presented with several partial epileptic seizures with or without secondary generalisation due to brain metastasis. He received palliative chemotherapy consisting of orally administered tegafur/uracil/calcium folinate. To control the epilepsy, he was prescribed phenytoin. The patient developed inefficacy of the antiepileptic treatment due to the drug interaction of folinic acid from his chemotherapy regimen and phenytoin. The metabolic interaction between folinic or folic acid and phenytoin causing a decrease in phenytoin plasma levels is a well-known pharmacokinetic interaction (We read with attention the paper of Brickell The interaction between phenytoin and folinic acid is common, but less recognised in new treatment regimens, as shown in this case. Based on the unpredictable effects of folinic acid with or without 5-FU on phenytoin, we would like to advocate the use of antiepileptic drugs without interaction with chemotherapeutics, like gabapentin or levetiracetam, in patients with cerebral metastasis suffering epileptic seizures.


Drosophila embryo to shed light on the mechanism by which the L1-type cell adhesion molecule Neuroglian regulates axon growth.Cell adhesion molecules have long been implicated in the regulation of axon growth, but the precise cellular roles played by individual cell adhesion molecules and the molecular basis for their action are still not well understood. We have used the sensory system of the neuroglian mutant embryos. Axons stalled at a variety of positions along their normal trajectory, but most commonly in the periphery some distance along the peripheral nerve. All lateral and dorsal cluster sensory neurons examined, except for the dorsal cluster neuron dbd, showed stalling. Sensory axons were never seen to project along inappropriate pathways in neuroglian mutants and stalled axons showed normal patterns of fasciculation within nerves. The growth cones of stalled axons possessed a simple morphology, similar to their appearance in wild-type embryos when advancing along nerves. Driving expression of the wild-type form of Neuroglian in sensory neurons alone rescued the neuroglian mutant phenotype of both pioneering and follower neurons. A partial rescue was achieved by expressing the Neuroglian extracellular domain. Over/mis-expression of Neuroglian in all neurons, oenocytes or trachea had no apparent effect on sensory axon growth.We have found a highly penetrant sensory axon stalling phenotype in We conclude that Neuroglian is necessary to maintain axon advance along axonal substrates, but is not required for initiation of axon outgrowth, axon fasciculation or recognition of correct growth substrates. Expression of Neuroglian in sensory neurons alone is sufficient to promote axon advance and the intracellular region of the molecule is largely dispensable for this function. It is unlikely, therefore, that Nrg acts as a molecular 'clutch' to couple adhesion of F-actin within the growth cone to the extracellular substrate. Rather, we suggest that Neuroglian mediates sensory axon advance by promoting adhesion of the surface of the growth cone to its substrate. Our finding that stalling of a pioneer sensory neuron is rescued by driving Neuroglian in sensory neurons alone may suggest that Neuroglian can act in a heterophilic fashion. The fly genome contains only one member of this family, nrg, which undergoes alternative splicing to generate two transcripts [167, which is expressed widely on epidermis, muscle, glia and trachea, and a longer form, Nrg180, which is restricted to neurons. The two isoforms have identical extracellular and transmembrane regions and differ only in the carboxy-terminal ends of their cytoplasmic domains.The genetic tools available in nscripts ,7. TheseDrosophila. Null nrg mutants display a highly penetrant motor axon stalling phenotype in the body wall of the embryo [Nrg has been shown to regulate axon growth in the peripheral nervous system (PNS) during both embryonic and post-embryonic development of e embryo . This phDrosophila [Nrg is also involved in guidance of bristle mechanosensory (BM) and ocellar pioneer (OP) axons in the adult ocellar sensory system (OSS) of osophila ,9. Ablat167, which has an identical extracellular region to Nrg180, fails to rescue OSS axon defects in the nrg LOF mutant [There are conflicting data as to whether the intracellular domain of Nrg is necessary to mediate the effects of this molecule on axon growth. Expressing an artificial, glycosylphosphatidylinisotol (GPI)-anchored form of Nrg in wing sensory neurons and their epidermal substrate can generate the same gain-of-function phenotypes as the full length protein , suggestF mutant . This reDrosophila embryo as a model to further explore these issues. The cellular events underlying the peripheral growth of individual sensory axons are well characterized [nrg mutants, rather than studying adult phenotypes of hypomorphic or temperature sensitive mutants. This should provide us with a more reliable assessment of normal gene function. Our analysis shows that Nrg is essential for maintenance of sensory axon advance in the embryo, but not for correct axon fasciculation or pathfinding. The requirement for Neuroglian function in embryonic sensory neurons appears, therefore, to be less complex than in other contexts in Drosophila. Nrg expression is required in sensory neurons alone and the extracellular region of the molecule is at least partially able to perform this function. Our findings may also suggest that Nrg can promote sensory axon advance by a mechanism involving heterophilic molecular interactions.Clearly, many questions remain concerning the mechanism of action of Nrg in the regulation of axon growth. We have chosen the sensory nervous system of the cterized ,12 and scterized -15, provIn this study, we have focused on the growth of axons from the dorsal and lateral sensory neuron clusters in abdominal segments A1 to A7. During normal development, sensory axons contact other sensory neurons or their axons shortly after emerging from the neuron cell body. The dorsal cluster axon fascicle initially grows internally to the transverse connective branch of the trachea, turns ventrally, joins the intersegmental nerve (ISN) in the dorsal body wall region and continues growing ventrally towards the central nervous system (CNS) ,14. It hnrg LOF mutant embryos by DiI labeling. While there is some normal variability in the timing of axon growth, all of the identified sensory neurons we examined have entered the neuropile and begun to form terminal arborizations by mid-stage 16 in 100% of wild-type embryos . Sensory neurons showed a highly penetrant axon stalling defect in nrg mutant embryos. Axons were judged to have stalled if they had not entered the neuropile. Stalled axons consistently showed a simple morphology, terminating in the nerve with a simple, club-shaped growth cone with, at most, one or two projecting filopodia  a Figure . Cell bos Figure .Axon stalling occurred at a variety of positions along the normal axon trajectory. Axons seldom stalled before entry into the ISN: Figure nrg14 and nrg17 LOF alleles (Table P < 0.01) in nrg14/Y embryos  as seen in wild-type embryos , presum180 on sensory neuron cell bodies and sensory and motor axons as well as the expression of Nrg167 on glial cells and trachea [During embryogenesis, Nrg shows a widespread pattern of expression. Of particular relevance to its role in the regulation of sensory axon growth is the expression of Nrg180 using the P0163-GAL4 driver in the nrg14 null mutant background. We focused on the two neurons for which we had multiple fills in nrg14 mutant embryos – lch5-1 and lch5-5. To confirm that expression of Nrg180 had been specifically restored in sensory neurons, we carried out BP104 immunohistochemistry (which recognizes an epitope specific to Nrg180) on nrg14/Y;P0163-GAL4/UAS-nrg180 embryos. Positive staining was seen in sensory neurons, including the lch5s and their axons, but not in central neurons  than in the nrg14 mutant  than for the nrg14 mutant . This may suggest that the nrgGPI construct is not as effective in promoting axon advance as the full length nrg construct, although it is also possible that differences in the level of expression of the two constructs could account for this apparent difference in rescuing ability.The Drosophila wing [180 in the embryonic PNS using either the P0163-GAL4 driver or the pan-neural elav-GAL4 driver. Confocal microscope observations on wild-type and P0163-GAL4 X UAS-nrg180 embryos stained with BP104 under the same conditions confirmed that Nrg180 was expressed at higher levels in the transgenic embryos than in wild-type embryos . mAb49c4 immunohistochemistry, which labels chordotonal neurons specifically, did reveal an aberrant dorsal misprojection of lch5 axons but only in 1/85 hemisegments of elav-GAL4 X UAS-nrg180 embryos.Neither form of overexpression resulted in sensory axon stalling as determined by DiI injections  or the trachea (with the btl-GAL4 driver) – cell types that lie in the vicinity of the lch5 neurons and their axons [Misexpression of Nrgir axons ,15 – hasDrosophila embryo to determine the consequences of complete ablation of Nrg function on sensory axon growth. nrg LOF mutants show a highly penetrant sensory axon stalling phenotype. This defect is displayed by all neurons examined, except for the dorsal cluster neuron, dbd, which apparently leads other dorsal cluster axons as it grows along the ISN into the CNS. The fact that this pioneering neuron is unaffected by loss of nrg function may suggest that Nrg is necessary for axon advance only along axonal substrates. This idea is supported by our observation that lateral cluster axons in nrg mutants almost always advance successfully along their initial non-neuronal growth substrate, the spiracular branch of the trachea, and stall only after they have subsequently joined the ISN.We have used the embryonic sensory system of the nrg mutants generally advance for some distance along nerves before stalling, Nrg is apparently required to maintain axon growth along axonal substrates, rather than to initiate it. Most axon stalls occur in the periphery, but their locations are quite variable and do not obviously coincide with particular structures, such as nerve branches, pointing to a stochastic component in the stalling process.As sensory axons in nrg mutants are always found in their correct nerve pathways. The absence of axon misprojection phenotypes shows that Nrg is not necessary for recognition of correct growth substrates by sensory axons.Stalled sensory axons in nrg mutants fasciculate normally with other axons. The simple growth cone morphology of the stalled axons – club-shaped with few filopodia – is similar to that seen in wild-type sensory axons when they are growing along nerves. These observations show that Nrg is not necessary for axon-axon fasciculation in this system. The Ig-CAM Fasciclin2 (Fas2) is a key regulator of motor axon fasciculation in the Drosophila embryo [fas2 LOF mutants and in embryos over/misexpressing Fas2 in sensory neurons and their growth substrates . Whether Nrg acts redundantly with some other CAM to mediate sensory axon fasciculation remains to be determined.We also observed that sensory axons in a embryo  and alsoa embryo . Howevera embryo . In addinrg mutant embryos are similar to those reported for motor axons, although motor axons stall at a later stage of growth, close to their synaptic targets, the body wall muscles, and unlike sensory axons, some motor axons misproject in nrg mutants [nrg LOF phenotype in the embryonic sensory system is much more stereotypic than in the adult OSS [The sensory axon defects seen in  mutants . The nrgdult OSS . This dinrg mutants, such as motor axon stalling [While Nrg is expressed in both sensory axons and several of their growth substrates, including trachea and motor axons, our mutant rescue experiments demonstrate that expression in sensory axons alone is sufficient to mediate their advance. It is unlikely, therefore, that the sensory axon defects are secondary to other known morphological defects in stalling  or failustalling .in vitro [in vivo. We have shown that expression of Nrg in sensory neurons completely rescues the axon stalling phenotype of lch5-1 seen in nrg mutants. In wild-type embryos and in nrg mutants, lch5-1 is the pioneer for the lateral cluster fascicle and associates with motor axons, rather than dorsal cluster sensory axons as it grows towards the CNS. Our lch5-1 mutant rescue findings might suggest, therefore, that Nrg is acting in a heterophilic fashion to promote advance of this axon. This conclusion rests upon the assumption that, in nrg mutant embryos in which the lch5-1 stall phenotype has been rescued, the lch5-1 growth cone does not employ the dorsal sensory axons as a growth substrate as it advances along the ISN. Observations of the dynamics of lch5-1 growth cone activity, and its association with sensory and motor axons in the ISN as it advances along the nerve in these rescued embryos, would help to resolve this question.An important, unresolved question is whether the effects of Nrg on axon growth are mediated by homophilic interactions between Nrg on the growth cone and its substrate or whether heterophilic interactions are involved. Substrate-bound Nrg has been shown to promote neurite extension from Nrg-expressing neurons in vitro , but it in vitro models used in most vertebrate studies to date do not accurately reflect the in vivo functions of L1-type CAMs. Alternatively, Nrg, the Drosophila L1 homologue, may function differently to vertebrate L1-type CAMs in promoting axon growth.Our mutant rescue experiments show that the intracellular region of Nrg is largely dispensable for its role in promoting sensory axon advance. It has long been known that Nrg-mediated cell-cell adhesion does not require the intracellular region of the molecule . Howevercis with another receptor or CAM, which in turn is coupled to the cytoskeleton of the growth cone. A genetic screen, using the sensory axon stalling phenotype described in our study as an assay, would be one way of identifying such a Nrg-interacting molecule.Our finding that the intracellular region of Nrg is not essential for its ability to promote sensory axon advance raises the question of how Nrg-mediated adhesion of the growth cone to its substrate is coupled to retrograde F-actin flow. Current models assume that such a coupling provides the motive force for growth cone advance. One possibility is that Nrg interacts in Drosophila embryo by promoting adhesion between the surface of the growth cone and its axonal substrates. This adhesive interaction is likely to involve a heterophilic interaction between Nrg on the growth cone and some other, as yet unidentified molecule, on the substrate. Intracellular signaling by the Nrg molecule does not appear to be essential for its function in this context. Our findings for Nrg are mirrored by the recent discovery that the adhesive, rather than the signaling activity, of another CAM, Drosophila N-cadherin, is essential for its role in target selection of photoreceptor afferents [Taken together, our results suggest that Nrg may mediate sensory axon advance along nerves in the fferents .nrg mutant alleles were used, nrg14 and nrg17  and an artificial Nrg isoform (UAS-nrgGPI), in which the extracellular region of Nrg is membrane-tethered with a GPI-moiety [GAL4 lines were used to drive expression of these UAS lines: P0163-GAL4, which drives expression in all sensory neurons and their support cells [elav-GAL4 line [sal-GAL4, which drives expression in oenocyte precursors [btl-GAL4, which drives expression in trachea [lacZ-marked balancer chromosomes to enable genotyping of embryos by fluorescence microscopy or anti-β-galactosidase immunohistochemistry. Crosses were conducted at 25°C with the exception of UAS-GAL4 crosses, which were carried out at either 25°C or 29°C.All stocks were raised on standard cornmeal and sugar medium. Two h Figure . Two UASI-moiety . The folrt cells ; the panAL4 line ; sal-GALecursors ; and btl trachea . All mut180, BP104 [Eggs were collected for 6 hours at 25°C then placed at 18°C for an additional 16 hours, or collected overnight at 25°C on apple juice-agar plates with yeast paste. Embryos were staged on the basis of gut morphology  and leng0, BP104  (mouse I0, BP104  (mouse ISensory neurons were individually stained in intact embryos by juxtacellular injection of DiI, as previously described ,17. FollBM: Bristle mechanosensory; CAM: Cell adhesion molecule; CNS: Central nervous system; DSHB: Developmental Studies Hybridoma Bank; EGFR: Epidermal growth factor receptor; Fas2: Fasciclin2; GPI: Glycosylphosphatidylinisotol; ISN: Intersegmental nerve; LC: Longitudinal connectives; LOF: Loss-of-function; Nrg: Neuroglian; OP: Ocellar pioneer; OSS: Ocellar sensory system; PNS: Peripheral nervous system; SN: Segmental nerve; v': Ventral'.The authors declare that they have no competing interests.nrg mutants and mutant rescue experiments. VM and MCS jointly carried out phenotypic analysis of effects of Nrg overexpression on lch5 morphology. JR and DJM carried out phenotypic analysis of dbd morphology in nrg mutants. PMW conceived of the study, participated in its design and coordination and was largely responsible for writing the manuscript. All authors read and approved the final manuscript.VM and EM jointly carried out neuron dye filling and phenotypic analysis for all neuron types, except dbd, in


We describe popliteal arterial adventitial cystic disease which causes intermittent claudication in a young athletic man, with atypical manifestation, without loss of foot pulses on knee flexion nor murmur in the popliteal fossa. The findings obtained from Magnetic Resonance Imaging were non-diagnostic. The diagnosis resulted from Echo-Doppler ultrasonography along with peak exercise testing. Ultrasonography also provided useful physiopathological informations suggesting that a popliteal artery adventitial cyst can become symptomatic if muscle exertion increases fluid pressure within the cyst, enough to cause hemodynamically significant endoluminal stenosis. Rapid diagnosis is essential to prevent progressive claudication threatening limb viability. To guarantee this professional sportsman a reliable and durable outcome, instead of less aggressive management, we resected the involved arterial segment and interposed an autologous saphenous-vein graft. Popliteal arterial adventitial cystic disease is a rare disorder causing intermittent claudication that is well described in young adults, and was first reported by Atkins and Key  in a 40-In patients with advanced disease, the diagnosis generally rests on evidence of segmental popliteal artery stenosis or occlusion without other popliteal artery disease. Traditional angiography or more recently gadolinium-enhanced magnetic resonance imaging (MRI) angiographic scans disclose an eccentric popliteal artery narrowing known as the "scimitar" sign. In some cases, stenosis is visible only on images obtained during knee hyperflexion.Although the precise etiology remains unknown, now the most accepted mechanism involves cystic degeneration causing vessel stenosis or occlusion without superimposed thrombosis. Increased pressure from the liquid within the cyst is thought to cause popliteal stenosis through an "intermittent" mechanism induced by physical exercise.In the 34-year-old man we report here, since MRI angiography failed to visualize the cyst clearly, the definitive diagnosis of popliteal arterial adventitial cystic disease came from the ECHO-Doppler ultrasound examination.We describe here the case of a 34-year-old man, a professional football player in excellent physical conditions, with no clinical history of importance except traumatic rupture of the anterior cruciate ligament, the right in 1985 and the left in 1995, treated with reconstruction using the central third of the patellar tendon. The patient presented with the abrupt onset of intermittent claudication in the right calf after walking on the flat for 300–400 meters.An Echo-Doppler ultrasound examination disclosed a suspected "small aneurysm" or "popliteal adventitial cyst" but no popliteal artery disease was seen on MRI angiography even on images obtained during forced knee flexion.On the patient's admission to our vascular service, an Echo-Doppler ultrasound examination, obtained during hyperflexion, disclosed no peripheral perfusion deficit. On treadmill exercise testing, after 500 mt. the incline was increased from 10% to 15% and the speed from 6 km/h to 8.6 km/h. About 19 minutes later (about 1900 mt.) the patient began to experience pain in the right foot and, after exercising for a further 250 mt. with the incline at 12% and the speed at 7 km/h, he complained of cramp-like pain in the right calf. At peak force, and during cramp, the Echo-Doppler ultrasonogram showed an accentuated stenosis that involved the lumen of the right popliteal artery and corresponded to an increase in cystic volume. These changes were not discernible during baseline testing.   and the Particularly remarkable features in this report are the Echo-Doppler ultrasonographic findings clearly documenting the pathophysiological mechanism through which the popliteal artery adventitial cyst became symptomatic. Although previous investigators -8 descriIn cases like this reported here, ultrasounds examination can be more accurate than imaging techniques such as CT scan or MRI; Moreover ultrasounds are less expensive, widely available and free of ionsiong radiations.This possible mechanism is supported from observations, unlike the case we report, describing cysts communicating with the synovial structures in the knee. Numerous reports describing treated Baker's cysts, that ultimately recurred, underline the need for meticulous surgical dissection of the cyst away from the adjacent joint structures in these cases and, also, in similar cases of cysts developing contiguously with the hip joint . ArguingOur decision to manage our patient's cyst by resecting the involved arterial segment and interposing an autologous saphenous vein graft, rather than undertaking CT- or ultrasound-guided drainage or surgical evacuation, depended primarily on the need to guarantee a young adult with a professional sports career a reliable and durable outcome.In spite of their rare occurrence, popliteal artery adventitial cysts are a disabling disease especially because they typically affect young otherwise healthy athletic adults. The diagnosis is usually reached by MRI angiography at rest. Even though arterial stenosis can become symptomatic and therefore visible on ultrasonography only after maximal prolonged exercise testing, ultrasonography is diagnostically far more reliable than more traditional imaging modalities. Popliteal arterial adventitial cystic disease should be managed surgically without delay, before the artery occludes, and radical surgery is essential to avoid the known risks of recurrence.The authors declare that they have no competing interests.MT and NS permormed the surgery and participated in the design of the study, LR and MM conceived the study and wrote the manuscript, FC gave orthopaedic contribution to the operation, CM performed the ultrasound test with VF.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal


Important attributes of 3D brain cortex segmentation algorithms include robustness, accuracy, computational efficiency, and facilitation of user interaction, yet few algorithms incorporate all of these traits. Manual segmentation is highly accurate but tedious and laborious. Most automatic techniques, while less demanding on the user, are much less accurate. It would be useful to employ a fast automatic segmentation procedure to do most of the work but still allow an expert user to interactively guide the segmentation to ensure an accurate final result. We propose a novel 3D brain cortex segmentation procedure utilizing dual-front active contours which minimize image-based energies in a manner that yields flexibly global minimizers based on active regions. Region-based information and boundary-based information may be combined flexibly in the evolution potentials for accurate segmentation results. The resulting scheme is not only more robust but much faster and allows the user to guide the final segmentation through simple mouse clicks which add extra seed points. Due to the flexibly global nature of the dual-front evolution model, single mouse clicks yield corrections to the segmentation that extend far beyond their initial locations, thus minimizing the user effort. Results on 15 simulated and 20 real 3D brain images demonstrate the robustness, accuracy, and speed of our scheme compared with other methods.


The mitochondrial oxidative phosphorylation (OXPHOS) is critical for energy (ATP) production in eukaryotic cells. It was previously shown that genes coding for mitochondrial proteins involved in energy production co-express at the RNA level. Because the OXPHOS enzymes are multimeric complexes, we tested the hypothesis that genes coding for components of specific complexes are also co-regulated at the transcriptional level, and share common regulatory elements in their promoters.In silico analysis of homologous promoter sequences in mammals identified the likely core promoter elements for most genes encoding OXPHOS subunits/assembly factors. The results included a significant abundance of previously identified sites , as well as several sites that had not been previously detected. Although we identified patterns that correlated to OXPHOS gene expression, we did not detect an OXPHOS complex-specific arrangement of transcription factor binding sites within the core promoter that could explain the tight co-expression of these functionally related genes.We observed for the first time that, not only OXPHOS genes as a group co-express, but there is a co-expression of genes within each of the five OXPHOS enzyme complexes, showing a higher degree of complexity in gene co-regulation. This study mapped the core promoters of most OXPHOS related genes and provided an example of gene expression regulation based on the final protein arrangement within a linear metabolic pathway. The oxidative phosphorylation system (OXPHOS) is responsible for 90% of adenosine triphosphate (ATP) production in a respiring cell. Five multi-subunit complexes, the respiratory chain complexes, and two additional electron carriers, coenzyme Q10 and cytochrome c, participate in OXPHOS to generate ATP -4.Microarray data have been widely used to study gene co-expression by studying genes with similar expression patterns across a set of samples. Over the past few years, several lines of evidence suggest that co-expressed genes  amongst different expression experiments) are likely to encode proteins that participate in the same metabolic pathway, form a common structural complex, or might be regulated by the same mechanism ,6.In eukaryotes, the regulatory mechanisms underlying co-regulation of multiple genes is extremely complex . At the Energetic demands vary substantially between different cells and tissues of an organism. For example, in mammals, the mitochondrial content of cardiac myocytes or brown adipose cells is very high compared to skeletal muscle fibers type IIb. In general, the energy demand of a specific tissue correlates with the level of expression of genes encoding components of the OXPHOS system . The mecThere are diverse regulatory mechanisms that may underlie co-expression. However, most studies have been devoted to the identification of proteins that regulate the transcription of nuclear-encoded mitochondrial genes as well as factors regulating mitochondrial transcription. Recent evidence points to both transcription factors (TFs) and transcriptional coactivators as important players in the regulation of mitochondrial biogenesis. DNA-binding TFs including nuclear-respiratory factor 1 and 2  estrogen related receptor alpha (ERRA), Sp1, ying yang 1 (YY1), CREB and E-box factors have all been implicated in the expression of some nuclear-coded respiratory chain subunits, among other genes -15. TranThe main conclusion we can draw from the above is that the expression of OXPHOS genes can be modulated at the transcriptional level. There is mounting evidence to show that TFs and coactivators, particularly PGC-1α, are able to cooperatively alter the expression of OXPHOS genes. Although OXPHOS gene expression can be regulated at different levels, gene expression is likely to be a major determinant for coordinated expression. Therefore, understanding the complete set of TFs and coactivators that regulate OXPHOS gene expression should yield insights into the control of OXPHOS gene expression regulation. Although significant progress has been made in this respect, a complete list of factors regulating the expression of all OXPHOS genes remains incomplete, and how these regulators are integrated under physiological conditions is poorly understood.In this study, OXPHOS gene co-expression across different tissues in humans and mice was confirmed in an independent large microarray dataset. Detailed analyses showed that subunits of individual complexes co-express preferentially among each other than with subunits of other complexes. In addition, phylogenetic footprinting across human, mouse and rat, helped to define each OXPHOS gene core promoter, and resulted in a comprehensive list of factors that could participate in their transcriptional control.From various genome wide expression studies, a significant group of OXPHOS genes appears to be co-expressed under various physiological conditions in several species. To identify which particular OXPHOS genes are co-expressed in different conditions/species, we obtained the complete microarray dataset of human and mouse from the Genomics Institute of the Novartis Research Foundation (GNF) tissue compendium. Using custom-designed whole-genome gene expression arrays from panels of mRNAs derived from 79 human and 61 mouse tissues and cell types performed in duplicates, this compendium evaluates the relative expression of 44,775 human and 36,182 mouse transcripts . AnalyziIn order to study co-expression of OXPHOS genes among all the human and mouse "mitochondrial genelists", an unsupervised hierarchical cluster analysis was performed. This approach was used to visualize relationships among the expression patterns of OXPHOS genes and other groups of genes. Figure In both mice and humans, the hierarchical tree cluster analysis resulted in a cluster of genes enriched in OXPHOS genes. These results confirm previous observations that a significant number of OXPHOS genes share a common pattern of gene expression across different tissues and this co-expression is conserved across different species. Other gene groups that were significantly co-expressed among different tissues correspond to genes coding for fatty acid biosynthesis pathway, oxidative stress response, mitochondrial translation machinery, mitochondrial chaperone activity, glycolysis and calcium binding. Tissue clustering showed that tissues with high or low expression of OXPHOS genes clustered together. Tissues with known high energy consumption such as heart, skeletal muscle, kidney and liver displayed increased expression of OXPHOS genes compared to tissues with lower energy demand such as skin and lung. This pattern of expression was observed for other mitochondrial genes as well, which suggests that tissues with high energy demand have not only an increase in the expression of energy generating genes but also of other mitochondrial components. These proteins may be necessary to structurally and metabolically sustain increased energy production.The above study suggests that a significant number of OXPHOS subunits co-express among different tissues in both mouse and humans. Next, we studied if particular members of each respiratory complex co-express among each other preferentially over subunits/assembly factors of other OXPHOS complexes. To address this issue, an "OXPHOS correlation test" was designed (please refer to the methods section for a full explanation of this test). Briefly, a similarity matrix was created for all OXPHOS genes Figure . Next, tTable This analysis showed that although OXPHOS subunits co-express among themselves within and across species, gene members coding for each multimeric complex also co-express preferentially with members of the same complex. This novel observation suggests that there is a higher degree of complexity in OXPHOS gene regulation than had been so far detected.From the above study it is apparent that OXPHOS genes co-express at a tissue level. This raises the hypothesis that these genes have a common co-regulatory mechanism that allows co-expression among different tissues. Previous studies suggest that transcriptional initiation is the most common form of gene regulation of OXPHOS genes . HoweverRespiration is a vital process in essentially every eukaryotic cell, thus OXPHOS genes are considered housekeeping genes and are expressed in all cell types. In vertebrates, CpG islands are usually associated with several housekeeping gene promoters. We tested for the presence of CpG islands in all human OXPHOS subunit promoters. Overall, CpG islands are present in the promoter regions of 83 out of the 101 human OXPHOS genes analyzed. Since during the course of evolution GC dinucleotides are usually lost, this analysis suggests that there is a high evolutionary pressure for sequence conservation of OXPHOS promoter sequences, and these conserved stretches of DNA probably reflect important transcriptional regulatory elements, and possible nucleosome positioning that would contribute to transcriptional regulation .A hallmark of several OXPHOS promoters studied until now is the absence of a typical TATA box adjacent to the TSS. These promoters are called TATA-less . The TATFrom our studies as well as others previously published, the fact that OXPHOS genes are coregulated is clear. Therefore, with the assumption that functionally important TFBS are conserved across evolution, searching for conserved TFBS in OXPHOS promoters would probably result in important information on the molecular basis of OXPHOS transcriptional regulation.To search for conserved TFBS, phylogenetic footprinting was performed on human, mouse and rat orthologous promoters for all OXPHOS genes. Using DialignTF , orthologue promoter sequences were aligned and strings of short-bp similarity  were searched against a database of known TFBS. The most abundant TFBS families identified belong to TFs previously linked to transcription of some OXPHOS genes: ETSF (Human and murine ETS1 factors) which includes NRF2, SP1F (GC-Box factors SP1/GC), NRF1, EREF (Estrogen response elements) which includes ERRA TFBS, CREB (cAMP-responsive element binding proteins) and YY1F (Activator/repressor binding to transcription initiation site). We also identified TFs known to regulate housekeeping genes, such as EGRF (EGR/nerve growth factor induced protein C & related factors), ZBPF (Zinc binding protein factors) and EBOX (E-box binding factors) among others. Please refer to supplementary material . NRF1, ERRA, YY1 and CREB TFBSs showed a statistically significant enrichment in OXPHOS co-expressing gene promoters in both human and mouse tissues Figure . A previOverall, these results show that phylogenetic footprinting resulted in the detection of several TFBS that are enriched in OXPHOS gene promoters. Some TFBS that displayed a high abundance in OXPHOS gene promoters, like SP1F, did not show an association with OXPHOS gene expression, which suggests that they might participate in non-specialized basal transcriptional initiation of these genes. The vast majority of the OXPHOS promoters analyzed have not been characterized previously. Therefore, these results confirm that the unbiased computational approach chosen does lead to biologically relevant information.A fundamental question in a bioinformatics approach to promoter analysis is to determine which of the predicted TFBSs are biologically relevant. In the above study, the presence of a TFBS conserved across human, mouse and rat was taken as a guide to answer this question. In addition, the relative position of each conserved TFBS to the corresponding TSS can also be analyzed. Previous studies have relied on this approach, and found that TFBS that cluster relative to the TSS  have a high likelihood of being biologically significant .Following this idea, the positional bias of each TFBS relative to the TSS was recorded. Although the analysis covered 500 bp surrounding the TSS (500 bp upstream and downstream), the conserved TFBS identified by phylogenetic footprinting preferentially occur within ~150 bases of the TSS in all three organisms, consistent with the likelihood of these TFBS being involved in transcription initiation Figure . PreviouTaking a closer look around the TSS it became apparent that YY1F TFBS are preferentially conserved at the TSS or around 10 bp of it, suggesting a strong role in transcription initiation of OXPHOS genes Figure . On the in silico approach.In summary, there seems to be a clear positional bias around the TSS for the most abundant TFBS identified by phylogenetic footprinting. In particular, known OXPHOS related TFs, NRF1, NRF2 and YY1F (except ERRA) all cluster within ~150 bp of the TSS. This suggests that most conserved TFBS identified for OXPHOS promoters are probably transcriptionally functional in humans, mouse and rat, and raises the possibility that TFBS identified outside of this region might not be functional for the promoters of these ubiquitously expressed genes studied. These results further support the biological relevance of our From previous studies, most TFs do not have a preferred orientation of binding in order to play a role in transcription initiation. To test this in OXPHOS promoters, TFBS orientation was recorded for all conserved TFBS detected in all three species. YY1F and EGRF families of TFBS showed a statistically significant p < 0.05) bias towards one strand of DNA  was normalized to its median across all chips or arrays. These normalization steps enabled comparisons of relative change in gene expression levels between experiments. Duplicate arrays were then averaged and considered as one.The human and mouse expression atlas was obtained from GNF ,28. ThisData was filtered for mitochondrial genes. A complete list of human mitochondrial genes was obtained from two online compilations, which contain data obtained by several proteomic approaches in an effort to obtain the complete list of mitochondrial genes. MitoRES  providedHierarchical clustering analysis was performed on the "mitochondrial compilation" genelists using Pearson correlation similarity. Genespring software package was used to create a graphical view of the data.For the OXPHOS correlation test (explained bellow), two more datasets were used. The U74A mouse tissue compendium from GNF  and the To investigate if OXPHOS subunits co-express preferentially with subunits of the same complex than with subunits of other complexes, a "correlation similarity" test was designed. For each microarray dataset, a similarity matrix was made. Pearson's correlation was used to measure the pair-wise similarity between the expression profiles among all OXPHOS genes in the matrix Figure . If one Because some complexes have very few nuclear coded genes, the above analysis did not pass our statistical test for complexes with few subunits . To bypass this restriction, we grouped the results of the OXPHOS correlation test for all probesets from the four datasets before applying the statistical test.Genomatix  and the The presence of CpG islands was assessed by the UCSC Genome Browser built-in application . The algOrthologous promoter sequences from human, mouse and rat were aligned using DiAlignTF . This application is able to align and identify conserved TFBSs in orthologous promoters. DiAlignTF is a DNA alignment software that constructs alignments from gap-free pairs of similar segments of sequences. Therefore, the program is especially suited to detect short local similarities, characteristic of short TFBS, in otherwise completely unrelated sequences. DialignTF was used with its default parameters using the Genomatix Matrix Family Library Version 5.0 (January 2005) and the orthologous promoters for each gene. This alignment is often called phylogenetic footprinting. For this analysis, 98 OXPHOS orthologous promoters  as well as 134 other gene promoters participating in several mitochondrial pathways were examined.Because several transcription factors bind similar TFBSs, Genomatix groups these into TFBS families. For example, NRF2 binding site is similar to other ETS family members, and therefore, ETSF (ETS family) contains NRF2 binding site. Since NRF2 has been implicated in the expression of some OXPHOS subunits, for this analysis, promoters were analyzed for the presence of both ETSF (family) and NRF2 (single factor) binding sites. On the other hand, NRF1 has its unique TFBS and therefore is not part of any family of TFBS.Conserved families of TFBSs in all orthologue promoters were retrieved. The presence of at least one copy of a conserved TFBS family in the aligned promoter sequences was examined. The presence of more than one copy of the same TFBS family was not recorded. Binding sites for the transcription factor families V$SF1F, V$RORA and V$ERER (Genomatix nomenclature) are very similar and therefore often overlap . V$ERER Human, mouse and rat promoters were used to search all OXPHOS promoters for the presence of a TATA box. If a TATA box protein binding site (V$TBPF) was found to be conserved within 100 bp of the TSS of the aligned promoters, it was considered as a likely functional TATA box. If no V$TBPF binding site was present the genes were considered as being TATA-less .To test if TFBSs identified by phylogenetic footprinting are enriched in OXPHOS gene promoters over random mitochondrial gene promoters, a statistical test was designed. This test was performed with all promoters analyzed by phylogenetic footprinting using DialignTF default parameters n = 235). An ordered list of all genes was generated using the Pearson correlation to the expression pattern of the average expression pattern of OXPHOS genes  of each conserved TFBS analyzed above was determined. A nonparametric Sign test was used to determine if there was a preference of TF binding orientation. TFBS orientation depends on the Genomatix library of position weight matrices (PWM).ATP: Adenosine-5'-triphosphateChIP: chromatin immunoprecipitationCOX: Cytochrome c Oxidase (a.k.a. Complex IV)CREB: cAMP response element binding proteinCV: Pearson's correlation valuecyt c: Cytochrome cERRA: Estrogen related receptor alphaETC: electron transport chainGNF: Genomics Institute of the Novartis Research FoundationHIF: hypoxia inducible factorKCN: potassium cyanideMELAS: Mitochondrial myopathy Encephalopathy Lactic acidosis and Stroke-like episodesmiRNA: micro RNAmRNA: messenger RNAmtDNA: Mitochondrial DNAnDNA: nuclear DNANRF1: Nuclear respiratory factor 1NRF2: nuclear respiratory factor 2OXPHOS: : Oxidative Phosphorylation SystemPGC-1: Peroxisome-proliferator activated gamma coactivator-1ROS : reactive oxygen speciesRT-PCR: reverse transcriptase polymerase chain reactionSDH: succinate dehydrogenaseTCA cycle: tricarboxylic acid cycleTF: Transcription factorTFAM: mitochondrial transcription factor ATFBS: Transcription factor binding sitetRNA: transfer RNATSS: transcription start siteYY1: ying yang 1OXPHOS: Oxidative Phosphorylation SystemCvW designed, executed, performed the statistical analysis and wrote the manuscript. CTM designed and wrote the manuscript. Both authors read and approved the final manuscript.Ordered list of genes used for mRNA profile in figure Click here for fileOXPHOS mRNA correlation test. The accompanying tables correspond to the similarity matrix used to perform the OXPHOS correlation test. The Pearson's correlation value (CV) for each pair of genes is shown. The values shown are a measure of the pair-wise similarity between the expression profiles among each pair of OXPHOS gene in the matrix.Click here for fileExample of phylogenetic footprinting. The figure shows how the phylogenetic footprinting approach is performed for the COX 6A gene.Click here for filePhylogenetic footprinting of Complex I core promoters. Core Promoters of Complex I subunits/related genes identified by phylogenetic footprinting. The conserved regions are shown in color codes, which correspond to specific Transcription Factor Binding Sites (TFBS).Click here for filePhylogenetic footprinting of Complex II core promoters. Core Promoters of Complex II subunits/related genes identified by phylogenetic footprinting. The conserved regions are shown in color codes, which correspond to specific Transcription Factor Binding Sites (TFBS).Click here for filePhylogenetic footprinting of Complex III core promoters. Core Promoters of Complex III subunits/related genes identified by phylogenetic footprinting. The conserved regions are shown in color codes, which correspond to specific Transcription Factor Binding Sites (TFBS).Click here for filePhylogenetic footprinting of Complex IV core promoters. Core Promoters of Complex IV subunits/related genes identified by phylogenetic footprinting. The conserved regions are shown in color codes, which correspond to specific Transcription Factor Binding Sites (TFBS).Click here for filePhylogenetic footprinting of Complex V core promoters. Core Promoters of Complex V subunits/related genes identified by phylogenetic footprinting. The conserved regions are shown in color codes, which correspond to specific Transcription Factor Binding Sites (TFBS).Click here for filePhylogenetic footprinting of promoters. Phylogenetic footprinting results of all promoters analyzed obtained by DialignTF . Sheet "conserved TFBS" contains the list of all promoters analyzes with the conserved TFBS obtained using the default paramenters of DialignTF. Sheet "Summary" contains a Summary of the data obtained using default parameters of DialignTF. Sheet "conserved TFBS SEL" contains data obtained using DialignTF in default and SEL parameters. Sheet "SEL" indicates the TFBS selected and the parameters used for the analysis in SEL mode. Sheet "Matrix Family Library" contains all Genomatix Matrix families (TFBS families) used in this study.Click here for filePrediction of core promoters vs. experimentally identified. The table compares promoter elements identified experimentally (left column) with the predicted in this study (right column).Click here for fileData used to perform the statistical analysis of TFBS abundance. These spreadsheets show the data used to perform the statistical analysis of TFBS abundance. Human and Mouse GNF microarray data were used to obtain the Pearson correlation values (CV) of each gene to an OXPHOS average gene . Genes with a higher CV value are those whose expression pattern was most similar to OXPHOS subunits. Each gene is also annotated for the presence of each TFBS in its promoter.Click here for file


Peptidergic neurons store and secrete the contents of large dense core vesicles (LDCVs) from axon terminals and from dendrites. Secretion of peptides requires a highly regulated exocytotic mechanism, plus coordinated synthesis and transport of LDCVs to their sites of release. Although these trafficking events are critical to function, little is known regarding the dynamic behavior of LDCVs and the mechanisms by which their transport is regulated. Sensory neurons also package opiate receptors in peptide-containing LDCVs, which is thought to be important in pain sensation. Since peptide granules cannot be refilled locally after their contents are secreted, it is particularly important to understand how neurons support regulated release of peptides.+ is rapid and terminates within 15 minutes, while phorbol ester stimulation of secretion is maintained over a longer period. The data demonstrate fundamental differences between LDCV dynamics in axons and growth cones under basal conditions.A vector encoding soluble peptidylglycine α-hydroxylating monooxygenase fused to green fluorescent protein was constructed to address these questions in cultured primary peptidergic neurons of the trigeminal ganglion using time lapse confocal microscopy. The time course of release differs with secretagogue; the secretory response to depolarization with KUnder basal conditions, LDCVs move faster away from the soma than toward the soma, but fewer LDCVs travel anterograde than retrograde. Stimulation decreased average anterograde velocity and increases granule pausing. Data from antibody uptake, quantification of enzyme secretion and appearance of pHluorin fluorescence demonstrate distributed release of peptides all along the axon, not just at terminals. Communication within the nervous system relies on the stimulus-dependent release of neurotransmitters from vesicles. Neurons contain two vesicle classes competent for regulated exocytosis: small synaptic vesicles (SSVs) and large dense core vesicles . Different stimuli regulate secretion from SSVs and LDCVs. Unlike neuropeptide-containing LDCVs, SSVs contain low molecular weight neurotransmitters synthesized in the cytosol . Newly sIn contrast to synaptic vesicles, LDCVs contain peptides synthesized as inactive precursors into the endoplasmic reticulum and transported to the trans-Golgi network, to be packaged into immature LDCVs and transported down the axon . These LLittle is known regarding the dynamic behavior of LDCVs and the mechanisms by which their transport is regulated in neurons. Most previous studies have utilized neuroendocrine cells such as PC12 or chromaffin cells, which preludes the analysis of events that occur during transport along long axons ,10 Compa+ methyl-2-aminoethanesulfonic acid (TES), 10 mM mannitol, and 1% Triton X-100, pH 7.4 (TMT) with protease inhibitors [125I labeled α-N-acetyl-Tyr-Val-Gly as substrate [Cells were rinsed 3 times with pre-warmed L-15, collections were made of basal secretion, followed by stimulation with 1 μM PMA or 50 mM KCl in L-15  made by mixing L-15 with isotonic 150 mM KCl. When indicated, temperature was used as a variable and cells were kept at either room temperature or above 30°C for the duration of the experiment. Medium was centrifuged to remove non-adherent cells, and protease inhibitors added. Cells were harvested in 20 mM Na-hibitors , extractStatistical analyses were performed using SPSS (PASW 17.0).In order to investigate the dynamic behavior of LDCVs in living neurons, we utilized a primary culture system of trigeminal ganglion neurons Figure . In our The soluble secretory protein PHM-GFP is efficiently targeted to LDCVs in endocrine cells  and was ++-dependent manner. Application of phorbol myristate acetate (PMA) activates Protein Kinase C, which phosphorylates various components of the SNARE complex as well as ion channels, causing secretion [Localization of PHM-GFP to the regulated secretory pathway was confirmed by quantifying basal and stimulated secretion. We chose two secretagogues with different mechanisms of action for our studies. An increase in KCl depolarizes the cells, potentially triggering action potential initiation, and opens voltage-gated calcium channels to increase secretion in a Caecretion . In ordeecretion -25. AlthTo select the time period over which LDCV motion was evaluated, we quantified secretion of PHM-GFP for 5 to 30 minutes following application of basal medium or medium containing KCl or phorbol ester at 37°C Figure . StimulaThe dynamics of LDCVs were first characterized under basal conditions. Dissociated trigeminal ganglion neurons were transfected with vector encoding PHM-GFP, and imaging experiments performed 24-48 hours later. This timing was chosen because process outgrowth occurred rapidly and growth cones were abundant. The short time made it possible to identify the cell soma associated with the growth cone chosen for imaging and distinguish anterograde from retrograde trafficking. Growth cones of small diameter neurons containing fluorescent LDCVs were identified, and the entire growth cone and the axonal segment immediately preceding it were imaged. Selective photobleaching of axonal segments was performed to facilitate LDCV tracking. Z-stacks were acquired continuously and maximum-intensity projections were generated for tracking analysis. Trajectories were generated for all tracked LDCVs based on the coordinates at each point in time; sample trajectories for two LDCVs that displayed different types of behavior are shown Figure .-4 μm2/sec; for the dithering LDCV (#235), D = 95 × 10-4 μm2/sec. For LDCVs diffusing freely in the cytosol, D = 10 × 10-4 μm2/sec [LDCV #219 moved in a rapid and directed manner without changing direction, while LDCV #235 demonstrated slow, dithering motion, with frequent changes in direction. The tracking protocol was validated by manually comparing the trajectories with the actual LDCV motion in several time lapse image sets. In order to distinguish directed from diffusive motion, we used the method of Steyer and Almers to calculate diffusion constants (D)  for thes μm2/sec . Polysty μm2/sec ,28. ThusVelocity, linearity, and direction were quantified for basal analysis of LDCVs in axons compared to growth cones. Velocity data of identified LDCV trajectories in growth cones and distal portions of axons were pooled from 6 cells each. Calculation of velocity was done as an average for the entire trajectory of each LDCV analyzed. The distribution of binned data is shown in the histogram Figure , with thLDCV trajectories were further analyzed to determine the distribution of linearity. Linearity was calculated by comparing how close the actual path was to a straight line representing the distance between the first and last point, so that a maximum linearity score is 1.00. As expected, the cumulative plot showed that LDCV tracks in axons were more linear than LDCV tracks in growth cones  Figure .The net direction of LDCV movement was determined based on the average angle of each trajectory. A score of 0 was assigned for a net angle representing retrograde movement (toward the cell body), and a score of 1 for movement in the anterograde direction (directed toward the ending). Cumulative directionality scores for LDCVs in axons and growth cones did not differ significantly Figure . In growThe lack of directionality in growth cones as compared to axons could be related to detachment of motor proteins upon entry into growth cones, and to a prevalence of actin over microtubules. We tested whether an intact cytoskeleton were required for efficient LDCV transport by pharmacological disruption of filamentous actin with cytochalasin B. Filamentous actin was visualized using a fluorescent phalloidin conjugate, verifying that cytochalasin treatment resulted in the rearrangement of F-actin into puncta Figure , red; mi-4 μm2/sec). Neither drug had a significant effect on overall LDCV velocity. When linearity was analyzed  in the trajectory using individual LDCV coordinates. The histogram in Figure The overall effect of secretagogue on instantaneous direction is displayed as an average for each treatment condition Figure . Under bNext, we analyzed the relationship between direction and velocity for each time interval Figure . LDCVs aTo be fully or partially secreted, a LDCV must pause or stop, at least momentarily ,30. We tMore frequent stopping of LDCVs in axons is suggestive of docking and fusion events occurring along the length of the axon. To assess this possibility we used an antibody to the luminal domain of a LDCV membrane enzyme, PAM. Internalized antibody was subsequently visualized in fixed cells with a Cy3 conjugated secondary antibody. To increase the signal obtained, PAM-GFP was expressed in trigeminal neurons; PAM-GFP was visible in tubulovesicular structures in the cell soma and in vesicular structures throughout axonal processes Figure , left. USince this approach did not permit direct visualization of fusion events in real time, we constructed a vector encoding PHM fused to pHluorin, a pH sensitive mutant of GFP . Neurons4Cl was added to the medium in order to visualize the total LDCV population containing PHM-pHluorin  occurred in axons, not in endings/growth cones.In order to detect release of PHM-pHluorin, a horizontal line was placed through the center of the axon and growth cone Figure , right, n Figure , right. in vitro trafficking studies.Our analysis of LDCV dynamics took a novel approach by utilizing primary neurons specialized to produce high levels of neuropeptides. Secretion of peptides such as CGRP and substance P from dorsal root and trigeminal ganglion neurons is an essential component of the transmission of nociceptive stimuli and contributes to peripheral inflammatory responses. In addition, these LDCVs deliver opiate receptors to the cell surface and may be involved in opiate receptor internalization as well . Unlike LDCV mobility is essential for sustaining the secretory response at distant sites of release. New LDCVs must come from the cell soma, the only site at which preproneuropeptides are synthesized and packaged into LDCVs. Since trigeminal ganglion neurons released 15% of their cell content during a 30 minute exposure to PMA or KCl, we hypothesized that these secretagogues would alter LDCV transport. We first compared LDCV transport in axons and growth cones. Differences were expected since long distance movements must occur in axons and secretion is generally assumed to occur at terminals.Under basal conditions, LDCVs in axons moved with greater average velocities than LDCVs in growth cones. LDCVs moving with average velocities greater than 0.1 μm/second were seen only in axons; these rapidly moving LDCVs accounted for only ~20% of the total LDCVs observed. Trajectories were more linear in axons than in growth cones, reflecting the influence of axonal geometry. We focused our analysis on the distal segments of axons, the region immediately preceding a growth cone. To our surprise, under basal conditions, LDCVs in this region were slightly more likely to move back toward the cell soma than into the adjacent growth cone.2+ stimulation increased the mobility of the subset of atrial natriuretic factor-GFP LDCVs located immediately adjacent to the plasma membrane, suggesting that the releasable pool might be expanded by making more LDCVs available to refill it [Drosophila neuromuscular junction following depletion [The most significant effect of secretagogue on average LDCV dynamics was its effect on trajectory linearity. Both PMA and KCl stimulation resulted in more linear LDCV paths in axons, suggestive of motor proteins staying attached longer. The average velocity of transported LDCVs was unaffected by stimulation with KCl or PMA, consistent with the lack of effect of KCl on orphanin FQ containing LDCV velocity in NS20Y cells . Averageefill it . Mobilizefill it . This liefill it . These aefill it . Using wepletion . They coepletion .Our spinning-disk confocal microscopy approach allows us to look over a prolonged observation time and to track LDCVs that move up and down with respect to the dish surface, as they traverse the axon. Our average LDCV velocity measurements included 7 to over 30 images (10 to 50 seconds of tracking). The fact that LDCVs paused and then began to move again is not reflected in these average velocity measurements. Since LDCVs must stop at least momentarily before they can be secreted, we quantified the movements made by each LDCV from one image to the image taken 1.7 seconds later; these are designated instantaneous velocities and include all LDCVs anywhere within the region of interest. LDCVs whose centers remained in the same pixel were scored as paused. Secretion of LDCV content would result in diminution or complete loss of signal and termination of that track. Both partial secretion of a docked LDCV, which has been shown to occur ,14, and en passant terminals of Drosophila motorneurons are replenished via capture of retrogradely transiting LDCVs, not by arrival of LDCVs from the soma [When we analyzed the effects of PMA and KCl on instantaneous direction and velocity, significant differences were observed. KCl both increased the fraction of LDCVs undergoing anterograde transport and decreased the fraction undergoing retrograde transport. PMA did not alter the anterograde/retrograde distribution of LDCVs. Interestingly, KCl but not PMA increased instantaneous velocity in the retrograde direction, while both KCl and PMA decreased instantaneous velocity in the anterograde direction. Neuropeptides stored in the the soma . These dMost LDCVs were found to travel less than 0.2 μm/sec. Fast axonal transport is typically defined as microtubule-kinesin-dynein dependent movement at 20 mm/d or faster 0.2-4.5 μm/sec), and slower transport within axons has long been recognized to be bidirectional . We foun.5 μm/secThe vast majority of previous studies of LDCV dynamics and exocytosis utilized neuroendocrine cell lines as model systems, and nearly all were performed at room temperature. This has included chromaffin cells, which are unpolarized ,31,36,41in vivo has yet to be determined. It will be interesting in the future to correlate activity- dependent LDCV dynamics and secretion with physiological functions.Our findings that LDCVs containing PAM-GFP or PHM-pHluorin undergo fusion along axons as well as at growth cone/endings in trigeminal ganglion neurons are consistent with a role for non-synaptic peptide release. In fact, a plausible interpretation of Figures In the peptidergic neurons of the trigeminal ganglion, LDCVs are transported away from the cell soma (anterograde) at a significantly faster rate than those traveling towards the cell soma (retrograde) under basal conditions. Anterograde LDCV velocity is slowed following depolarization by KCl or stimulation with PMA; both methods of stimulation increase granule pausing and LDCV exocytosis. Consistent with this, secretagogue stimulates the uptake of antibody that recognizes the luminal domain of a LDCV membrane protein and also stimulates the distributed appearance of a pHluorin-tagged LDCV content protein along the axon.JAS developed the culture system, synthesized the vectors used, acquired the images and performed the enzyme assays. WAM assisted with spinning disk confocal imaging, and AEC with line scan imaging. REM and BAE conceived of the study, assisted with performing experiments and did much of the data analysis. All authors participated in writing the manuscript, read and approved the final manuscript.


A select group of patients with upper tract urothelial carcinoma may be appropriate candidates for minimally invasive management. Organ-preserving endoscopic procedures may be appropriate for patients with an inability to tolerate major surgery, solitary kidney, bilateral disease, poor renal function, small tumor burden, low-grade disease, or carcinoma in situ. We review the published literature on the use of topical treatment for upper tract urothelial carcinoma and provide our approach to treatment in the office setting. Nephroureterectomywith open excision of bladder cuff remains the standard of care fororgan-confined upper tract urothelial cancer in patients with a normalcontralateral kidney. Laparoscopic surgery has broadened the population ofpatients who are able to tolerate this surgery; however, due to concerns forpreservation of renal function or inability to tolerate surgery, a selectedgroup of patients may be suitable candidates for less-invasive management withendoscopic approach. Organ-preservingendoscopic procedures may be appropriate for patients with a solitary kidney,bilateral disease, poor renal function, or inability to tolerate major surgery , 2. In aThe role ofintravesical immunotherapy in non-muscle-invasive urothelial cancer of thebladder has been firmly established , 5. A vaPatients must be properly selected for topical upper tract treatment. Upper tractabnormalities are often identified as a filling defect on delayed images of aCT urogram or IVP done for hematuria work-up or during selective upper tractwashings for a positive bladder cytology in the absence of any bladderpathology. Our operative evaluation for positive cytology includes evaluationof both the bladder and upper tract for source of positive cytology. Initially,a rigid cystoscope is introduced and the bladder is inspected. The bladder isdrained and a bladder wash with normal saline is obtained for cytology byrinsing the bladder several times. Cytology from the first kidney is obtainedusing normal saline washes through a 5 french open-ended ureteral catheter. Toreduce the chance of bladder contamination, positive pressure saline is infusedthrough the catheter up until the point in which it is introduced into theureter The catheter is advanced up into the renal pelvis (~25 cm) and two 5–7 ccsaline washes are obtained followed by additional washes at 20, 15, and 10 cm.The catheter is then readvanced up into the renal pelvis where the remainingfluid is aspirated and pooled together with the other washes. A retrogradepyelogram is then performed from the renal pelvis downwards as the catheter isremoved. The same procedure is then performed on the contralateral upper tract.It should be noted that special care must be taken to avoid contamination ofspecimens by using different ureteral catheters for each side. If anyabnormality is seen on retrograde pyelogram, then the upper tract should beevaluated with ureteroscopy with subsequent upper tract wash, brush biopsy, ortissue biopsy as deemed appropriate. The bladder should be inspected and fiverandom bladder biopsies taken in a stellate manner . An essentialcomponent of a complete evaluation is to obtain a separately labeled biopsyfrom the prostatic urethra, which can serve as a sanctuary site for urothelialcarcinoma.Patients treated with topical infusion therapy typically have either carcinoma in situor lesions that have been resected endoscopically. A mass in the collectingsystem or ureter identified on retrograde pyelogram or CT urogram prompts afocused operative evaluation. Small lesions may be amenable to endoscopictreatment (ureteroscopic or percutaneous) as discussed elsewhere in thisspecial issue. It is highly unlikely that topical treatment by itself woulderadicate radiographically visible disease. After primary treatment or thepresence of hematuria, BCG treatment should be delayed 2 to 4 weeks to allowthe urothelium to heal and decrease the likelihood of systemic side effects. Ofnote, in patients with positive cytology from both the bladder and upper tractwith no mass lesion, the status of the upper tract should be consideredinconclusive. In these cases, we generally start with intravesical treatment asthe positive upper tract cytology could be due to contamination from thebladder. Restaging after intravesical therapy is prudent to reevaluate theupper tracts. If the bladder is disease-free and the upper tract cytologycontinues to be positive, then this rules out the possibility of contaminationand should be treated accordingly.We use an office-based approach for placement of ureteral catheter(s) for upper tracttherapy. Flexible cystoscopy is performed, sometimes with oralnarcotic/benzodiazapine premedication or intravenous sedation  based on patient preference. The ureteral orifice is visualized andcannulated with a 0.018 inch angled glidewire that is passed to the renalpelvis. A 4 F whistle-tip catheter is then passed over the glidewire . Under dWe use a treatment regimen of low-dose BCG (one-third to one-tenth standard dose) plusinterferon-alpha-2b (50–100 million units) in 50 cc normal saline based on theeffectiveness of this combination in non-muscle-invasive bladder cancer [Patients receive weekly treatment for 6 sessions over 6 weeks. Then, 6 weeks following the last treatment, patients are restaged withbilateral upper tract washings and retrograde pyelograms, bladder washing, andrandom bladder biopsies along with prostatic urethral biopsy. If the resultsare negative, consideration is given to 3 future maintenance treatmentsstarting 6 weeks later. While there have been no published results on theefficacy of maintenance treatment, the addition of 3 weekly maintenance treatments is well establishedfor bladder CIS [The true benefit of topical therapy, either as a primarytreatment for carcinoma in situ or as an adjuvant for endoscopically treatedtumors, is difficult to assess based on the variance in the reportedliterature. In contrast to bladder cancer, which is relatively common, uppertract urothelial cancer is uncommon and, therefore, a single center is not ableto accrue significant numbers of patients for a prospective study.Additionally, heterogeneous groups of patients receive such therapy .Retrospective case reviews can also have methodological flaws. The goal is tomaximize the effectiveness of treatment while minimizing side effects andcomplications. Multiple different treatment regimens have been utilized withBCG being used most commonly . No randIn 1996, Yokogi et al. analyzed therapeutic outcomes of BCG perfusion therapy for upperurinary tract CIS in 8 renal units—5 through apercutaneous nephrostomy tube and 3 through a retrograde ureteral catheter . Follow- In contrast, Nishino et al. used BCG perfusion treatment (instilled weekly for 4or 8 weeks) to treat upper tract CIS via retrograde catheterization with eithera 6 French ureteral catheter or an 8 French indwelling double J ureteral stent [An indwelling ureteral stent was used to treat 11 patients with upper tract CIS as reportedby Nonomura et al. in 2000 . Reflux The efficacy of retrograde flow to the upper tract via an indwelling double Jureteral stent has been questioned. Yossepowitch et al. used performedcystograms with an indwelling stent in place and reported that retrograde flowoccurred in only 56% of patients. Additionally, the mean minimal intravesicalvolume to obtain reflux was 170 mL, which is higher than the typicallyinstilled treatment volume .Miyake et al. evaluated the efficacy of intrarenal BCG instillation for the treatment ofCIS of the upper urinary tract . SixteenIn the adjuvant setting after ureteroscopic tumor ablation, Patel and Fuchs in 1998reported on the use of topical BCG therapy  . At a meIn one of the largest series of topical upper tract treatment, Thalmann et al. (2002)retrospectively evaluated the results of BCG therapy for upper urinary tractdisease in patients not eligible for nephroureterectomy . Thirty-α (100 million units) [The data regarding supplemental interferon is not firmly established, validated, orwidely used for bladder cancer and certainly not for upper tract transitionalcell carcinoma. However, there have been two reports of its use in treatingupper tract urothelial carcinoma. We have reported on 15 patients  with upper tract urothelial cancer after endoscopic resection whoreceived 6 weekly adjuvant low-dose BCG (one-tenth standard dose) andinterferon-n units) , 15. Ninα2b (50 million units) via ureteral catheter.With a median follow-up of 24 months, 80% demonstrated a complete response,while 20% had a partial response . Theauthors reported thatthe treatment was well tolerated in the office setting and did not note anycomplications.In 2007, Katz et al. published their initial experience with upper tract BCG-IFN . A serieLess information is available on the use of mitomycin C topical treatment. In 1997,Keeley and Bagley reported on adjuvant mitomycin C  in 19 patients  for high volume,recurrent, or multifocal urothelial carcinoma [Due to confounding variables and heterogeneous treatment groups, direct comparisonsbetween treatment groups in a study can be difficult. One study in 1996 fromMartínez-Piñeiro et al. reported on a series of upper tract carcinoma in which26 patients received adjuvant supplemental topical therapy, and attempted tomake comparisons between different treatments . BCG andSimilarly, comparisons between treatment and nontreatment groups may be complicated byselection bias. In 2004, Palou et al. presented results of the percutaneousapproach to resection of upper urinary tract urothelial carcinoma in which 14and 5 patients received BCG and MMC instillations, respectively [An alternative experimental treatment regimen in refractory patients is sequentialgemcitabine and mitomycin C. Medication dosage is 1 gm gemcitabine in 50 cc phosphate-bufferednormal saline, then 40 mg mitomycin C in 40 cc sterile water. The gemcitabineis instilled followed by the mitomycin immediately afterward. We reported in2006 on a group of patients with treatment refractory non-muscle-invasivebladder cancer (8 of 37 had upper tract involvement . GemcitaWithout largeprospective or randomized data, the topical treatment of upper tract urothelialcancer is to some extent anecdotal. Topical treatment of upper tract urothelialcancer has a role in selected patients, who must be committed to closefollow-up because of the risk of recurrence and more concerning progression.The goal of treatment is to provide noninvasive, nephron-sparing treatmentwithout compromising oncologic outcomes. Treatment can be successfullyperformed in the setting of a busy urologist's office. Further study is neededto identify the best candidates for this treatment approach and to determine whichagents and schedule are most optimal. Our office-based approach to upper tracturothelial carcinoma provides the clinician with the framework to implementthis treatment in practice.