Purpose. We evaluated the ability of novel optical coherence tomography (OCT) parameters to predict postoperative best-corrected visual acuity (BCVA) in macula-off rhegmatogenous retinal detachment (RRD) eyes. Methods. We reviewed the medical records of 56 consecutive eyes with macula-off RRD. Clinical findings were analyzed including the relationship between preoperative OCT findings and 6-month postoperative BCVA. Results. Six-month postoperative BCVA was significantly correlated with preoperative findings including retinal height at the fovea, total and inner layer cross-sectional macular area within 2 mm of the fovea, and preoperative BCVA . Multiple regression analysis revealed that the duration of macular detachment and total cross-sectional macular area were independent factors predicting 6-month postoperative BCVA . Conclusions. Measuring preoperative total cross-sectional area of the macular layer within 2 mm of the fovea with OCT is a useful and objective way to predict postoperative visual outcome in eyes with macula-off RRD. Although remarkable progress has been achieved in the surgical treatment of eyes with rhegmatogenous retinal detachment (RRD), it is still difficult to predict postoperative visual outcomes when RRD includes a detached macula, known as macula-off RRD. Newly developed less-invasive surgical interventions, particularly 25-gauge microincision vitrectomy surgery (25GMIVS), have led to a very high initial reattachment rate for eyes with RRD, currently about 95% –5. HowevPhotoreceptor apoptosis has been reported to mainly occur within 3 days of RRD onset in experimental animal models , 8 and tWe hypothesized that preoperative macular volume reflected photoreceptor apoptosis and could thus predict postoperative outcomes in eyes with macula-off RRD. In order to test this hypothesis, we developed new OCT parameters that could provide a suitable evaluation of the macular structure and then determined the relationship between these parameters and postoperative visual outcomes in macula-off RRD eyes.We performed a retrospective analysis of the medical records of 56 consecutive eyes with macula-off RRD that underwent surgical intervention with a 25-gauge trocar cannula system or scleral buckling from January 2011 to February 2014 at Tohoku University Hospital. Eyes were included only if complete reattachment of the RRD was achieved after initial surgery. Eyes were excluded if they had prior vitreoretinal surgery, proliferative retinopathy, retinal vascular disease, chorioretinal atrophy, or high myopia (more than −10 diopters), if we could not obtain clear preoperative OCT measurements due to a bullous RRD or vitreous opacity, or if a single OCT scan could not capture the detached fovea and the retinal pigment epithelium layer. After the purpose and procedures of the operation were explained, informed consent was obtained from all patients. The procedures conformed to the tenets of the Declaration of Helsinki, and the study was approved by the Institutional Review Board of Tohoku University Graduate School of Medicine.μm area and was automatically calculated by the onboard OCT software. Our analysis of preoperative OCT parameters included retinal detachment (RD) height  [All patients underwent a complete ocular examination 6 months after surgery. Best-corrected visual acuity (BCVA) was measured preoperatively and 1 and 6 months postoperatively with the Landolt C visual acuity chart. Decimal acuity values were converted to logarithm of the minimal angle of resolution (log MAR) units. The detached macula was examined with spectral-domain (SD) OCT  in all patients preoperatively, and foveal thickness was also measured 1 and 6 months postoperatively. To evaluate the detached macula preoperatively, a 2 mm circle was manually centered on the surface of the fovea in a cross-sectional OCT macular image. The image of the macula within this circle was then manually segmented into three layers: the inner layer (nerve fiber layer and ganglion cell layer), middle layer (inner plexiform layer and inner nuclear layer), and outer layer (outer plexiform layer and outer nuclear layer) . All OCTal base)  and macut-test. Independent variables affecting 6-month postoperative BCVA were determined with multiple linear regression analysis. The significance level was set at P < 0.05.All statistical analysis was performed with JMP software . The correlation of 6-month postoperative BCVA to preoperative characteristics and operative, visual, and anatomical outcomes was determined with Spearman's rank correlation coefficient. Significant differences between preoperative and 1- and 6-month postoperative BCVA were determined with the paired P < 0.001, P < 0.001, and P < 0.001, resp.; P = 0.006, P < 0.001, and P = 0.001, resp.; P < 0.001 and P < 0.001, resp.). Multiple regression analysis revealed that the duration of the macular detachment and the total cross-sectional macular area were independent factors predicting 6-month postoperative BCVA . The cause of this discrepancy is unclear but may have been related to the instability of RD height, particularly in bullous RRD and particularly in older eyes, because the detached macula can more easily shift its position in the vitreous. It is difficult to accurately and reproducibly evaluate RD height in such eyes, leading us to speculate that preoperative RD height cannot be considered a reliable predictor of postoperative visual function in eyes with macula-off RRD.This study showed that RD height at the fovea, a measurement parameter used in a number of earlier studies, was associated with postoperative BCVA in a single regression analysis, confirming earlier reports , 12. ThiLimitations of this study included a relatively short follow-up time of 6 months, a relatively small sample size (about 60), and the omission of postoperative functional findings from standard automated perimetry or focal electroretinography. Additionally, although bullous RRD eyes are often seen in the clinic, the method described here cannot be used to predict postoperative outcomes in cases when OCT scans do not show the macula. Furthermore, to prevent bias in the results, it was necessary to omit the inner macular layer in the cross-sectional image from our multiple regression analysis, because the total and inner layer values were not independent, both being OCT findings and being closely correlated with 6-month postoperative BCVA. At first, we hypothesized that visual outcome would be associated with the area of the outer macular layer, as this contains the outer nuclear layer and the photoreceptor cells, but this hypothesis was not borne out by the data. It is unclear why this was so, but it may have been related to the susceptibility of the outer layer to intraretinal edema or undulation, which makes it difficult to obtain accurate measurements. Nevertheless, we believe our results show that simple OCT measurement of total cross-sectional area within 2 mm of the fovea is currently the most useful and objective way to predict postoperative visual outcomes in eyes with macula-off RRD, at least until technology to quickly evaluate macular volume in three dimensions becomes available for use in eyes with detached maculas. The usefulness of the measurement method described here would also be greatly enhanced by an OCT program to automatically measure cross-sectional macular area in eyes with macula-off RRD.In conclusion, OCT measurement of preoperative total cross-sectional area of the macular layer within 2 mm of the fovea is a useful and objective way to predict postoperative visual outcomes in eyes with macula-off RRD and was closely correlated with 6-month postoperative BCVA. Further investigation is needed to measure the macular volume and determine its relationship with visual outcomes, which could lead to the development of a new automatic OCT program.


Therapeutic hypothermia is an effective cytoprotectant and promising intervention shown to improve outcome in patients following cardiac arrest and neonatal hypoxia-ischemia. However, despite our clinical and experimental experiences, the protective molecular mechanisms of therapeutic hypothermia remain to be elucidated. Therefore, in this brief overview we discuss both the clinical evidence and molecular mechanisms of therapeutic hypothermia in order to provide further insights into this promising intervention. Clinicians have investigated the application of therapeutic hypothermia on the human body for centuries. Increasing clinical evidence from meta-analysis of large randomized controlled trials and experimental data advocates the induction of therapeutic hypothermia as a tool to achieve neuroprotection . ClinicaTo date, the strongest evidence for its efficacy exists for various clinical situations following cardiac arrest and neonatal hypoxia-ischemia. However, the effective application of hypothermia requires a thorough understanding of the injury mechanisms as well as its protective mechanisms. Therefore, we discuss in this brief overview both the clinical factors and molecular mechanisms of therapeutic hypothermia in order to provide further insights into this promising intervention.Early experiences with therapeutic hypothermia in the 1940s thru 1960s falsely assumed that the protective effects were only due to the temperature-dependent reduction in metabolism, which leads to lower oxygen and glucose demands . TherefoThree large multicenter randomized studies of newborn infants with hypoxic ischemic encephalopathy suggest a beneficial effect in this patient population. Gluckman et al. demonstrated an improved outcome that persisted at 18 months of life in term infants suffering from moderate neonatal encephalopathy who were subjected to head cooling  for 72 h . A seconCurrent investigations also include the Infant Cooling Evaluation (ICE) trial to investigate the effect of moderate whole-body hypothermia to 33.5°C for 72 h in newborns with HIE  and the Clinical issues regarding optimal target temperature, rate of cooling, duration of cooling, rate of rewarming, as well as optimal treatment window need further investigation. With the introduction of therapeutic hypothermia, it is important to differentiate between the ‘induction phase’ , when the temperature drops; the ‘maintenance phase’ , when the target temperature is achieved and maintained at the desired level; and the ‘rewarming phase’ , when the patient is slowly rewarmed back to normothermia.Hypothermia induces stress responses, such as shivering, that result in increased oxygen consumption and metabolic rate in non-sedated patients. Animal experiments suggest that the neuroprotective effects of hypothermia are negated if cooling is used on non-sedated animals ,22. OtheTherapeutic hypothermia-induced cellular protection against anoxic brain injury is a global process affecting multiple molecular and cellular mechanisms. Cooling results in a 6% to 10% decrease in cerebral metabolism for every 1°C reduction in body temperature . TherefoIschemic brain injury, reperfusion injury, and secondary brain damage are the three main categories of temperature-dependent injury processes that can be effectively mitigated by mild to moderate hypothermia. Due to the broad effects of hypothermia, it is more clinically effective than treatments that focus on blocking just one of these processes.Rate of cooling - rapid initiation of cooling immediately after injury resulted in better outcomes in animal studies. A concept summarized by the phrase ‘time is brain’.Duration of cooling - dependent of severity of injury and the time allotted to achieve target temperature.Rate of rewarming - slow rewarming is critical to not reactivate initial injury processes.Management and prevention of side effects.Variable factors such as type of injury (traumatic versus pure ischemia) and patient physiology  all contribute to the complicated injury processes. Although the window of opportunity to initiate hypothermic treatment as well as the duration of cooling to achieve full efficacy may vary, the success or failure of therapeutic hypothermia treatment is dependent on the following four key factors ,15:Rate Ischemia/reperfusion injuries can lead to cellular necrosis or apoptosis, also referred to as programmed cell death. The development of apoptosis is dependent on several cellular processes, including mitochondria dysfunction, activation of caspase enzymes, and other cellular energy metabolism disorders. Hypothermia has been observed to affect almost all of these injury processes, thereby preventing the initiation or interrupting the early stages of the apoptotic pathway . The win+ are dramatically increased, leading to both intracellular and extracellular acidosis and calcium (Ca2+) influx into the cells [2+, such as ATP-dependent Na+/K+ pumps and Na+, K+, and Ca+ channels [2+ eventually leads to mitochondria dysfunction and activation of the caspase-9 intrinsic apoptotic pathway. Data from experimental studies have proven that hypothermia has a beneficial impact on the ion pump dysfunction and reduces the influx of calcium into the cells, thereby decreasing neurotoxicity [Interrupted blood supply to the brain causes an immediate reduction in ATP and phosphocreatine, initiating a switch in intracellular metabolism to anaerobic glycolysis . As a rehe cells . To furtchannels . The exctoxicity ,29.Ischemia/reperfusion injury stimulates innate immune responses, which can lead to secondary brain injury. It triggers the release of pro-inflammatory cytokines  , the cheMild to moderate hypothermia has been shown to significantly reduce blood-brain barrier disruptions following ischemia-reperfusion injury by decreasing vascular permeability, resulting in decreased edema formation -41. Spec2−), peroxynitrite (NO2−), hydrogen peroxide (H2O2), and hydroxyl radicals (OH−) [The inflammatory response induced by ischemia/reperfusion injury usually coincides with the production of free oxygen radicals such as superoxide (Ols (OH−) . The extls (OH−) . Althougls (OH−) .Although hypothermia reduces cellular metabolism and down-regulates global protein synthesis in mammalian cells, a small subset of cold-shock proteins are induced by low temperature. RNA-binding motif protein 3 (RBM3), a member of the glycine-rich RNA-binding protein (GRP) family, is one of the first proteins synthesized in response to hypothermia  and hypoAlthough many studies on RBM3 have been focused on their regulation in non-neuronal cells in response to hypothermia and other stress factors , there iDrug-induced hypothermia may serve as an alternative to physical hypothermia, which can be technically tedious to apply and may cause serious side effects. Animal studies have shown that hypothermia is induced by synthetic cannabinoid CB1 agonists WIN55212-2 and HU-210. Intramuscular injection of WIN55212-2 induced rapid and prolonged hypothermia in a dose-dependent manner in rats , and HU-Temperature management continues to play an important role in the treatment of patients suffering from neurological injuries. Therapeutic hypothermia and it's proven ability to attenuate post-ischemic injury represents a quantum leap in the clinical setting , but mor


Cuculus canorus show little variation between individuals; yet, satellite tracks of 11 experimentally displaced adults revealed an unexpected flexibility in individual navigation responses. The birds compensated for the translocation to unfamiliar areas by travelling toward population-specific staging areas, demonstrating true navigation capabilities. Individual responses varied from travelling toward the first stopover in northern Europe to flying toward the Central-African winter grounds, the latter including several stopovers in unfamiliar areas. Apparently, the cuckoos possess spatial knowledge far beyond their population-specific flyway scale, and make individual decisions likely based on an assessment of perceived gain and cost of alternative route options.Migrating birds follow innate species-specific migration programs capable of guiding them along complex spatio-temporal routes, which may include several separate staging areas. Indeed, migration routes of common cuckoos  The complex innate spatio-temporal migration programs, capable of guiding migrant species thousands of kilometers, represents an evolutionary trade-off between species-specific resource needs and movement-related risks, resulting in varying migratory patterns across species and populations1681011We investigate the navigational capabilities in experienced individuals of a solitary, nocturnal migrant by experimentally displacing adult common cuckoos, a long-distance migrant with one of the most complex migrations known, using seven different staging sites between each breeding seasonShorter-distance (~100 km) directional responses in white-crowned sparrows suggest that experienced birds choose to travel directly toward their winter grounds after a long-distance displacement during the autumn migration1,17 = 64.8, P < 0.0001). The movements compensated for the displacement and were directed toward the post-breeding migration route normally followed by non-displaced birds. The pattern was similar for endpoint directions after 100 and 200 km .The initial response to displacement was movement toward east-northeast .Six displaced birds reached the migration route of the control birds after travelling 43–147 days . After crossing the desert, these four birds visited staging sites on latitudes corresponding to the Sahel stop over area  migration route. However, the birds stopped over in and navigated across the Sahel in latitudes 800–1100 km north of the spring routes followed by non-displaced birds between Central Africa and West Africa . First dThe navigational capability displayed by the adult cuckoos in this study indicates the existence of an extensive map sense reaching far outside the area normally traversed by individual birds. If the birds are relying on a gradient mapThis study was carried out in accordance with Guidelines to the use of wild birds in research of the Ornithological CouncilPosition estimates from ARGOS are assigned to a location quality class . After excluding all positions of class Z, we only used the highest quality position per transmission cycle unless the bird was in active movement, in which case all positions were included (following the procedure described in [14]). The movements of the 12 displaced birds were compared to the migration of eight non-displaced common cuckoos tagged in Denmark and southern Sweden (see [14] for a detailed description of this migration).We investigated the first, initial as well as the full, longer term navigational responses to the displacement. The initial response was defined as movements from the release site to the location after 15 days, potentially allowing for five transmission cycles. For comparison, we also used the first location after travelling 100 and 200 km. The full response was defined as movements from the release site to the first location on the normal migration route.We used a Rayleigh’s test to evaluate whether the direction to the end-point of initial responses were random. End-point directions of the initial responses were tested against specific directions (toward release location and stop over locations) using a confidence interval test, where the specific direction was considered different from the displaced group, if it was not included in the 95% confidence interval for the displaced group. The confidence interval was calculated as a bootstrap confidence interval around the mean direction in a von Misses distribution.We tested for differences among individuals in response to displacement for both first and full response by comparing directions of individual steps longer than 5 km using a Watson-Williams testStatistical tests were done in R 3.1.0www.movebank.org). Data are deposited in Movebank .


Central venous catheterization is of common practice in intensive care units; despite representing an essential device in various clinical circumstances, it represents a source of complications, sometimes even fatal, related to its management. We report the removal of a central venous catheter (CVC) that had been wrongly positioned through left internal jugular vein. The vein presented complete thrombosis at vascular ultrasonography. An echocardiogram performed 24 hours after CVC removal showed the presence, apparently unjustified, of microbubbles in right chambers of the heart. A neck-thorax CT scan showed the presence of air bubbles within the left internal jugular vein, left innominate vein, and left subclavian vein. A vascular ultrasonography, focused on venous catheter insertion site, disclosed the presence of a vein-to-dermis fistula, as portal of air entry. Only after air occlusive dressing, we documented echographic disappearance of air bubbles within the right cardiac cavity. This report emphasizes possible air entry even many hours after CVC removal, making it mandatory to perform 24–72-hour air occlusive dressing or, when inadequate, to perform a purse string. Venous air embolism is a well-known complication of venous catheterization in critically ill patients: it is generally related to insertion and removal procedures and daily management.Late air embolism after CVC removal is less known. We describe the case of a nonlethal air embolism 24 hours after removal of a malpositioned CVC, placed through left internal jugular vein, which was completely occluded by a thrombus.A 75-year-old woman was admitted to our intensive care unit for clinical monitoring after right parotid gland removal; she was diagnosed with a colliquative tumoral parotid gland mass in a peripheric hospital and then moved to the local otolaryngological surgical department to undergo surgery.Her past history included hypertensive cardiomiopathy, with episodes of heart failure, and atrial fibrillation.On admission to our unit she underwent a chest radiograph that showed a wrong positioning of the CVC : as shown in For these reasons and for the timing of catheterization (28 days before) we decided to remove it.The patient was mechanically ventilated with assisted mode, and she was placed in the head-down position before removing the catheter. Resistance was met during the removal of the catheter; after its dislodgement, local pressure using a gauze was performed to avoid bleeding. At inspection the catheter was whole, and a brownish scab on its distal tip compatible with a blood clot was found.2/FiO2 146).Afterwards she developed hemodynamic instability  that required adding inotropic support (dopamine 6 mcg/Kg/min) and deterioration of ventilation .


Methods. Eight mongrel dogs were randomly divided into 2 groups: half received an active TIIM pill and half received an identically sized sham capsule. After 24-hour fasting and transoral administration of the pill (active or sham), two force transducers (FT) were sutured onto the antral serosa at laparotomy. After closure, three standard cutaneous electrodes were placed on the abdomen, registering the transluminally emitted voltage. Thirty-minute baseline recordings were followed by pharmacological induction of gastric contractions using neostigmine IV and another 30-minute recording. Normalized one-minute baseline and post-neostigmine gastric motility indices (GMIs) were calculated and Pearson correlation coefficients (PCCs) between cutaneous and FT GMIs were obtained. Statistically significant GMI PCCs were seen in both baseline and post-neostigmine states. There were no significant GMI PCCs in the sham capsule test. Further chronic animal studies of this novel long-term gastric motility measurement technique are needed before testing it on humans.Transcutaneous intraluminal impedance measurement (TIIM) is a new method to cutaneously measure gastric contractions by assessing the attenuation dynamics of a small oscillating voltage emitted by a battery-powered ingestible capsule retained in the stomach. In the present study, we investigated whether TIIM can reliably assess gastric motility in acute canine models.  Distal postprandial gastric motility involves contractions that mechanically crush ingested food and mix it with secretions to prepare it for absorption, while maintaining an appropriate pressure gradient across the pyloric sphincter to regulate gastric emptying . The twoGastric function studies that can identify delayed gastric emptying include scintigraphy, C13 breath tests, wireless motility capsule tests, and combinations thereof . All theTranscutaneous intraluminal impedance measurement (TIIM) works by emitting a small high-frequency electrical signal from within the stomach, with parameters  chosen for their optimal transmission properties through smooth and abdominal muscles  and otheTIIM is a substantially different concept from standard electrogastrography (EGG), where cutaneous electrodes measure the intrinsic spontaneous electrical activity of the stomach. EGG has substantial drawbacks due to dynamic artifacts and lack of direct correlation with gastric muscular function, disease, or symptoms . ConversThe aim of the present study was to evaluate the effectiveness of the minimally invasive, ingestible, gastric-retentive TIIM capsule for measuring gastric motility in comparison to a sham gastric-retentive, ingestible capsule, with both being referenced to force transducers attached to the serosa of the stomach in acute canine models . This vaFor the present study, the TIIM miniature electronic oscillator was integrated into a gastric-retentive pill, which further lowers the already minimal impact of the catheter-based design . Each cuThis study was approved by the Life and Environmental Sciences Animal Care Committee, University of Calgary, Calgary, Alberta, Canada.−1 IV, Vetoquinol Canada, Lavaltrie, QC, Canada) and were continuously maintained on inhalant isoflurane and oxygen  with a vaporizer setting of 1%–3% until the end of the experiment, so that gastric motility was minimally affected. The anesthesia was chosen because it did not influence gastric neurotransmitters and as such would not affect gastric contractions [Experiments were performed on eight mongrel dogs (6 F) with a mean weight of 23.8 kg ± 3.3 kg, four of which were administered an active TIIM capsule, while the rest were given a deactivated (battery-removed) capsule. After 24 h fasting and 12 h water deprivation, each animal ingested transorally a single capsule as described above (TIIM or sham) with 500 cc of room-temperature water. The pill swelled to its maximum size in the stomach within 15 minutes after ingestion to dimensions exceeding 1.5 cm in any direction and subsequently was unable to pass the pyloric sphincter even when subjected to propulsive peristalsis. The animals then underwent induction with an intravenous injection of thiopental , and the serosa of the stomach was measured using an oscilloscope  to confirm the presence of an activated or deactivated pill. After this verification, two 90W24 force transducers , specifically designed for gastric motility monitoring, were surgically sutured to the serosal side of the antral stomach along the gastric axis . The firThe signals from the force transducers were amplified using a custom-designed multichannel bridge amplifier and digitized using a PCMCIA DAQ Card-AL-16XE-50 . The force transducer (FT) signals were monitored and analyzed with custom-designed signal processing and visualization software . Once the force transducers were in place, their functionality was verified mechanically by manual palpation of variable strength, and the offsets and gains were calibrated accordingly for maximal sensitivity. The intragastric position of the pill was then verified mechanically by palpating it to ensure that it had not been compromised during surgery.Following the FT implantation the abdomen was closed, and after appropriate skin cleaning and preparation, three pediatric ECG electrodes  were placed cutaneously over the stomach along the abdominal projection of the gastric axis, with a ground electrode positioned closer to the left hip of the animal . The posThe cutaneous electrodes were connected to a custom-designed multichannel electrogastrograph , which measured the surface electrical activity relative to ground. The cut-off frequencies of the bandpass filter of the EGG amplifier were set to the commonly used 0.03–0.1 Hz following the hypothesis that gastric motility signals in the animals will not exceed 6 cycles per minute (cpm)  and will−1, APP Pharmaceuticals, Schaumburg, IL) was administered intravenously as a smooth muscle stimulant to invoke contractions [Immediately after the experimental setup was completed , a baselractions . Thirty At the end of the experiments the animals were sacrificed by an IV injection of Euthanyl, 480 mg/4.5 kg . Subsequent retrieval of the expanded pill was performed in order to verify its retention within the stomach and confirm the presence of the signal in the active TIIM pills or the lack thereof in the inactive sham pills using an oscilloscope. The postadministration volume of each gastric-retentive pill was measured to quantify expansion dimensions.P < .05) [Since the cutaneous and the force transducer measurements were relativistic in nature, all measurements (2 cutaneous and 2 FT channels) were normalized with the maximal amplitude in 1-minute intervals becoming unity and the minimal set to zero. Thirty one-minute gastric motility indices  were calP < .05) .In addition, the frequency spectra of the signals were computed at 3.4-minute intervals with 64% overlap , and the frequencies of the dominant peaks per spectrum per modality  were recorded and averaged utilizing the custom-designed software package GAS 6.2.t-test [Furthermore, the dominant peaks of the frequency spectra for each measuring modality  were subjected to a comprehensive statistical analysis using the paired Student's t-test  to evaluPrior to the force transducer implantation, the electrical activity or lack thereof of the ingested pill was verified with an oscilloscope. In each animal the expanded pill was retrieved from the stomach at the end of the experiment, with an average postretrieval volume of 12.1 ± 0.4 mL and dimensions exceeding 1.5 cm in all directions, indicating that even the neostigmine-invoked contractions had not been able to propel the expanded gastric-retentive capsule beyond the pyloric sphincter.P < 0.05) correlations between the TIIM gastric motility indices and the FT gastric motility indices. In the case of the deactivated sham pill  [ < 0.05) . Sham re+-K+ ionic exchange at cellular level leading to cellular depolarization, which is a necessary but not sufficient condition for contractions to occur) [The clinical utility of gastric electrical measurements (EGG or bioimpedance) for assessing gastric motility has been previously shown to be limited , 14. Theo occur) . Gastrico occur) , 28. Theo occur) . Both ERIt is worth noting that although the aim of the present study was to further validate the TIIM technique, the sham pill study was fundamentally a direct comparison between gastric motility indices obtained via routine EGG and force transducers, since standard EGG bandpass filtering parameters (0.03–0.1 Hz) were used. In four animals no statistically significant correlation was observed in the motility indices between any of the channel pairs (FT-EGG) examined, further supporting our understanding that EGG cannot reliably monitor gastric motility due to its important and, in our opinion, unsurpassable limitation; the relatively low average electric-power of extracellular ERA-I and ERA-II preceding mechanically relevant gastric contractions dissipates intraluminally in an inconsistent and nonlinear fashion before reaching the abdominal skin surface and thus cannot be reliably recorded by traditional cutaneous EGG. With the electrically isolated, gastric-retentive pill-based design presented in this study, it is possible that environmental electrical phenomena such as capsule tumbling due to motion not associated with gastric contractions, static electricity, and external electromagnetic signals may affect the measurements in an ambulatory setting, particularly if the patient is mobile in an electromagnetically dynamic environment. In addition, in an ambulatory setting it is possible that abdominal muscle contractions and motion artifacts might also be registered. In such cases, adaptive filtering procedures  might heIt should be noted that in the case of the inactive sham study small noise artifacts appear dramatic since there is no 50 kHz carrier signal as a reference. In fact, these noise artifacts were much lower (10–100 times) than the measurements from the active TIIM study. However, since the signals were normalized (the highest peaks were equated to unity and the lowest were equated to zero) before visualization and subsequent presentation, electrostatic noises affected the cutaneous recordings of the sham TIIM signals, and they appeared as enlarged white noise. Unfortunately, we cannot avoid normalization, because there is no exact way to determine the basal impedance the TIIM electrical oscillator faces, which can also be dynamically changing, particularly in chronic studies of longer duration. Regardless, it should be noted that if the sham TIIM tracings were displayed on the same scale as the active TIIM figure, they would appear as virtually flat lines. In future long-term  chronic gastric motility studies utilizing TIIM, we do not perceive the normalization of the TIIM data to pose a significant problem, since the interdigestive motor complex and postprandial contractility would be clearly seen, particularly if a data logger marks the meal periods while recording the TIIM data.The next steps for TIIM involve the development of a portable data-logger to enable ambulatory measurements in long-term chronic tests, which can examine the effectiveness of the method in more clinically relevant settings, in dynamic, noise-ridden environments and during meals. Further long-term chronic animal studies (>24 h) should be considered, potentially evaluating gastric motility and gastric emptying using TIIM, before moving to chronic human trials.Transcutaneous intraluminal impedance measurement (TIIM) is a minimally invasive gastric motility monitoring technique which has now been validated in a sham-comparative study. Acute canine models confirmed that TIIM was able to measure gastric motility with comparable precision to force transducers implanted invasively to the serosa of the stomach.


For too long researchers have focused their attention on other most popular, sometimes clumsy or foggy forms of headache, overlooking this important and Apparently CH represents the most peculiar form among the Trigeminal Autonomic Cephalalgias (TACs). The unbearable periorbital side-locked pain is coupled by ipsilateral cranial autonomic symptoms  and causes a vast disability although the overall burden of this disease has not yet found a complete systematization.Especially its chronic form, characterized by more days with attacks than not in the year span, represents a strict minority  and hits 70.000 on a sample of 100 millions of people. Even if these numbers are relatively small the impact of this disease is destructive, thus CH represents a niche worth to be re-focused.Recently Chronic CH (CCH) refractory to whichever medical therapy (rCCH) has been systematized from a clinical point of view .The personal burden caused by CH, meant as loss of employment, homebound days and disability is higher in women than in men .CH lifetime prevalence is 1/1000 and over the past decades a progressive reduction of the male/female ratio has been reported, being now 2.1:1 . Even ifActually the current pathophysiological theory of CH is oriented towards a posterior hypothalamic dysfunction  but furtFor too many years sumatriptan, verapamil and corticosteroids have been the cornerstones of CH treatment ; we hopeOther approaches to chronic cluster headache are the new mini-invasive and non-invasive neuromodulation techniques. The European Headache Federation recommends caution in using these techniques  because ad hoc RCTs.Among these new approaches, Vagal Nerve Stimuation (VNS) and sphenopalatine ganglion stimulation (SPG) seem promising but still classified at Class IV evidence , 13; SPGLastly, patients’ education is another important topic to be re-considered: sleep pattern changes, alcoholic beverages, NO-derived cardiovascular drugs and phosphodiesterase inhibitors widely self-prescribed for erectile dysfunction trigger additional bouts during CH active phases . Furtherquasi-rare headache disorder.Thus we trust that this Open Thematic Series dedicated to Cluster Headache could thicken the attention on a famed and


Rapamycin has been shown to extend lifespan in rodent models, but the effects on metabolic health and function have been widely debated in both clinical and translational trials. Prior to rapamycin being used as a treatment to extend both lifespan and healthspan in the human population, it is vital to assess the side effects of the treatment on metabolic pathways in animal model systems, including a closely related non-human primate model. In this study, we found that long-term treatment of marmoset monkeys with orally-administered encapsulated rapamycin resulted in no overall effects on body weight and only a small decrease in fat mass over the first few months of treatment. Rapamycin treated subjects showed no overall changes in daily activity counts, blood lipids, or significant changes in glucose metabolism including oral glucose tolerance. Adipose tissue displayed no differences in gene expression of metabolic markers following treatment, while liver tissue exhibited suppressed G6Pase activity with increased PCK and GPI activity. Overall, the marmosets revealed only minor metabolic consequences of chronic treatment with rapamycin and this adds to the growing body of literature that suggests that chronic and/or intermittent rapamycin treatment results in improved health span and metabolic functioning. The marmosets offer an interesting alternative animal model for future intervention testing and translational modeling. Rapamycin has been found by multiple laboratories to extend mouse lifespan even when mice began receiving rapamycin relatively late in life at 20 months of age, or roughly the equivalent of 55 human years . In addiHowever, clinical administration of rapamycin has the potential for several side-effects include hyperlipidemia and hyperglycemia , raisingIn rodent models, monotherapy with rapamycin has largely, though not equivocally, been associated with impairment of glucose metabolism as measured in glucose tolerance tests. A notable exception is a study by Fang et al. suggesting that chronic treatment with rapamycin has a biphasic effect on glucose metabolism with short-term rapamycin treatment being detrimental to glucose metabolism whereas long-term (20 wk) treatment with rapamycin may promote an insulin-sensitive state in mice with a transition state in between . FurtherCallithrix jacchus) is a small monkey with a relatively short lifespan. Both its small size and associated shorter lifespan make this species a valuable nonhuman primate model for the study of aging and chronic disease [The common marmoset , resulting in no difference between control and rapamycin treated subjects at the end of the study as illustrated in Fig There were no significant differences between controls and rapamycin treated subjects and no effects of rapamycin treated subjects over time on body lean mass.There were no significant differences between controls and rapamycin treated subjects in food intake at two months post-dosing. In rapamycin-treated marmosets, there was a significant increase in food intake at month 5, over the pre-dosing food intake , as illustrated in Fig. In both control and rapamycin-treated subjects activity scored as accelerometer counts per hour declined significantly after the second month of dosing, as illustrated in Fig. There were no significant differences between controls and rapamycin-treated subjects in pre- versus post-dosing mean circulating triglyceride concentrations.There was, however considerable inter-individual variation in both baseline triglyceride concentration and in change over time, as illustrated in Fig. Three measures of glucose metabolic function were assessed: fasting blood glucose, QuickI index, and AUC. There were no significant changes in fasting glucose concentrations as illustrated in Fig. There were no differences between control and rapamycin treated subjects and no interaction effect on the glucose AUC. There were also no significant differences in post-dosing average glucose AUC in the rapamycin treated subjects through 8 months of dosing, as illustrated in Fig. The long-term administration of rapamycin in rodents has been associated with hyperglycemia caused in part by increased gluconeogenesis -29. In tBecause rapamycin modulated fat content of marmosets in the early periods of treatment, we assessed the potential modulation of effectors of lipolysis/lipogenesis in adipose. In visceral adipose samples, we found no significant effects on the phosphorylation or expression of any of these markers, suggesting little effect of rapamycin. However, at the time of sacrifice, rapamycin-treated marmosets were not significantly leaner than control animals. Due to limitations of the design of this study, we could not determine whether these effectors were altered by rapamycin at earlier time points when fat mass was reduced by treatment Fig. .Prior to assessing the effectiveness of rapamycin as an anti-aging treatment in humans, it is first necessary to elucidate the potential effects on long term health outcomes. In particular, there has been a great deal of controversy and inconsistent results in the clinical studies of rapamycin, which have highlighted the potential increased risk for metabolic defects such as hyperlipidemia and hyperglycemia that are consistent with increasing risk of cardiovascular disease and type 2 diabetes . HoweverMany have proposed that rapamycin is a mimic of calorie restriction which is the gold-standard for anti-aging intervention resulting in both extended life span and health span in many rodent models. Rapamycin has been shown to suppress mTOR activity, potentially decrease weight and fat mass, and extend healthspan and life span in a similar manner as calorie restriction -4. In maP. obesus treated with rapamycin display heightened hyperglycemia and increased insulin resistance in part by reducing pancreatic β-cell function. In this rodent model it was suggested that rapamycin exacerbated the pre-existing diabetic symptoms and metabolic disorder in high risk animals [One of the major reported risks of rapamycin administration clinically is the development of new-onset type 2 diabetes as shown in clinical studies of kidney transplant patients treated with rapamycin analogs , 34. How animals . Alterna animals , 41, 42; animals , and int animals , as well animals , 40-42.  animals , 9, 44.  animals ,37. In p animals ; while w animals , it may The increased risk of hyperglycemia with rapamycin treatment has been attributed to increased hepatic gluconeogenesis in rodent studies. In both mice and rats, rapamycin treatment significantly increases hepatic glucose production following injection with pyruvate and increases the expression of the gluconeogenic effectors like PCK1 and G6Pase , 46. In In addition to its well-known roles in cell survival and growth, recent studies have linked the mTOR signaling pathway with the regulation of lipid metabolism . HoweverTreatment with rapamycin as an intervention in the aging process for humans offers many possibilities but some studies have reported deleterious side effects that raise concern regarding the efficacy of this treatment. This study represents the first to examine the metabolic consequences of rapamycin dosing in healthy non-human primates. We have reported here evidence that long term rapamycin treatment at a dose that has been used in previous studies and reduces mTOR signaling in marmosets  does notCallithrix jacchus) housed at the Southwest National Primate Research Center. Basic husbandry and housing for this colony have been described previously [The subjects for this study were common marmosets (eviously . Thirteeeviously . Two paiMarmoset lean and fat mass was assessed monthly via quantitative magnetic resonance (QMR) imaging using an Echo MRI unit . UnsedatSubjects participated in a 2 day food intake trial once per month for the length of the trial . For theDaily activity patterns were assessed with the Mini actiwatch (CamNtech) which were placed in a marmoset pouch (Lomar) on a ferret harness (Petco). Subjects were gradually habituated to the ferret harnesses over the course of three weeks, increasing time in the harness incrementally throughout training until 24 hours in the harness had been achieved. The miniwatches are data loggers that batch data in 15 second epochs. For these trials animals were separated from each other within the cage and placed in harnesses with the actiwatch in the pouch secured across the back of the animal. Animals remained in the harness for 48 hours of data collection during which normal husbandry and feeding continued. At the end of the trial the animals were captured in transfer boxes and the harnesses were removed. Data was downloaded from the device. The activity counts from the first 15 minutes and last 15 minutes of the collection were removed from analysis as these represented handling and cage manipulation.Each month animals were fasted overnight and 2 ml of blood were drawn to assess circulating triglyceride concentrations, fasting glucose and insulin concentrations. Fasting glucose was determined immediately following the blood collection via glucometer. Blood was collected into serum separator tubes, spun and frozen in −80°C until further analysis. Triglyceride concentrations were assessed at the SNPRC clinical pathology lab. Samples were shipped to Wisconsin for analysis of insulin concentrations as described .Animals were fasted overnight prior to an oral glucose tolerance test , and plaTotal protein extracts were isolated from liver and visceral fat tissue that had been snapped frozen in liquid nitrogen after sacrifice and stored at −80°C until use. Protein extracts were homogenized in RIPA buffer with additional protease and phosphatase inhibitors , centrifuged at 14,000g at 4°C for 15 minutes, and then stored at −80°C until needed. Equal amounts of protein samples were separated electrophoretically by SDS-PAGE and then transferred to polyvinylidene difluoride membrane . Primary antibodies and their sources used in this study: adipose triglyceride lipase (ATGL), pyruvate carboxylase (PCB), glucose-6-phosphatase α (G6Pase), glucose-6-phosphate isomerase (GPI), and actin from Santa Cruz (Santa Cruz CA), peroxisome proliferator-activated receptor γ (PPARγ), phospho-pyruvate dehydrogenase kinase (p-PDK1), pyruvate dehydrogenase kinase (PDK1), phosphoenolpyruvate carboxykinase 1 (PCK1) from Cell Signaling (Beverly MA), sterol regulatory element-binding protein 1 (SREBP1) from Abcam (Cambridge MA) and deptor from Millipore with all alkaline phosphatase-conjugated secondary antibodies (anti-rabbit and anti-mouse) from Santa Cruz. Protein bands on immunoblots were detected using ECL reagent and analyzed using ImageJ.Variables of interest included body mass, fat mass, fat-free mass, 24 hour total actimeter counts, 24 hour caloric intake, triglyceride concentration, fasting glucose concentration, fasting insulin concentration, and glucose AUC following an oral glucose challenge. For each variable, the change in pre- Rapa dose value to post- Rapa dose value, measured following (one month) of dosing was compared for control subjects versus rapamycin-treated subjects in a two-way, repeated measures ANOVA. An additional repeated measures ANOVA was used to compare values within each rapamycin-treated subject over time for the entire dosing period. Comparisons of tissue protein activity were done using MANOVA with Bonferroni corrections. Analyses were conducted using GraphPad/Prism and SPSS 13.0.


Chloroplasts play a crucial role in sustaining life on earth. The availability of over 800 sequenced chloroplast genomes from a variety of land plants has enhanced our understanding of chloroplast biology, intracellular gene transfer, conservation, diversity, and the genetic basis by which chloroplast transgenes can be engineered to enhance plant agronomic traits or to produce high-value agricultural or biomedical products. In this review, we discuss the impact of chloroplast genome sequences on understanding the origins of economically important cultivated species and changes that have taken place during domestication. We also discuss the potential biotechnological applications of chloroplast genomes.The online version of this article (doi:10.1186/s13059-016-1004-2) contains supplementary material, which is available to authorized users. Chloroplasts are active metabolic centers that sustain life on earth by converting solar energy to carbohydrates through the process of photosynthesis and oxygen release. Although photosynthesis is often recognized as the key function of plastids, they also play vital roles in other aspects of plant physiology and development, including the synthesis of amino acids, nucleotides, fatty acids, phytohormones, vitamins and a plethora of metabolites, and the assimilation of sulfur and nitrogen. Metabolites that are synthesized in chloroplasts are important for plant interactions with their environment  and their defense against invading pathogens. So, chloroplasts serve as metabolic centers in cellular reactions to signals and respond via retrograde signaling , 2. The Nicotiana tabacum), was sequenced in 1986 [The advent of high-throughput sequencing technologies has facilitated rapid progress in the field of chloroplast genetics and genomics. Since the first chloroplast genome, from tobacco  facility was published recently . The lacOne of the important factors in the rapid advancement of the chloroplast genomics field is improvement in sequencing technologies. In studies conducted before the availability of high-throughput methods, isolated chloroplasts were used for the amplification of the entire chloroplast genome by rolling circle amplification –12. An aNandina and Platanus. Although multiple NGS platforms are available for chloroplast genome sequencing [The development of next-generation sequencing (NGS) methods provided scientists with faster and cheaper methods to sequence chloroplast genomes. Moore and colleagues  first requencing , Illuminquencing , 35, 36 quencing . Investiquencing . A thirdquencing –43. Its quencing , which fPotentilla micrantha, sequencing with the Illumina platform produced seven contigs covering only 90.59 % of the chloroplast genome; by contrast, using the PacBio platform with error correction, the entire genome was successfully assembled in a single contig [The low accuracy (~85 % of the raw data) of the long reads produced by the PacBio platform  can be ce contig .Cathaya argyrophylla) to 218 kb (Pelargonium), and is independent of nuclear genome size  regions Fig. . The chlHordeum vulgare) [Bambusa sp.) [Manihot esculenta) [Cicer arietinum) [clpP) occur in diverse plant species, including monocots (Poaceae), eudicots (Onagraceae and Oleaceae) and gymnosperms (Pinus) [Like the genes, the introns in land-plant chloroplast genomes are generally conserved, but the loss of introns within protein-coding genes has been reported in several plant species , includivulgare) , bamboo usa sp.) , cassavaculenta) , and chiietinum) . The proietinum) . The maj (Pinus) .At higher taxonomic levels (family level), protein-coding regions and conserved sequences of the chloroplast genome can be used for phylogenetic analysis and domestication studies . EarlierIn this section, we discuss several examples of comparisons of chloroplast genomes, within and between crop species, that have provided unique insight into evolutionary relationships among taxa. We also discuss the origin and geographic distribution of economically important species, as well as their adaptations to different climatic conditions and the use of genome information in their breeding and conservation.A key application of the chloroplast genome in agriculture is the identification of commercial cultivars and the determination of their purity. DNA barcodes derived from the chloroplast genome can be used to identify varieties and in the conservation of breeding resources. Success in breeding is determined by genetic compatibility and chloroplast genomes serve as a valuable tool for identifying plants that are likely to be closely related and, therefore, genetically compatible. Understanding the genetic relationships between cultivated crops and their wild relatives informs efforts to introduce specific advantageous traits into cultivated crops. In the section below, we discuss how chloroplast genomes have been used to elucidate the evolutionary relationships and domestication history of a few major crops and how this informs breeding programs.Odontoglossum ‘Violetta von Holm’, Odontoglossum ‘Margarete Holm’ and Odontocidium ‘Golden Gate’ are derived from the same female parent (Odontoglossum bictoniense) but phylogenetic analyses of ‘Violetta von Holm’ did not correlate with those of ‘Golden Gate’ or ‘Margarete Holm’ [The Orchidaceae is a large family that encompasses about 6–11 % of all angiosperms  and is ite Holm’ . A possite Holm’ . Althougte Holm’ . The usete Holm’ , 61.Sorghum bicolor), barley [Zea mays) [Triticum aestivum) [Secale cereale) [Oryza sativa) [http://www.ers.usda.gov/topics/crops/rice.aspx). The Oryza species are classified into ten genome types, including six diploids  and four allotetraploids . Attempts to clarify the evolutionary relationships between cultivated rice and its wild relatives remain contentious and inconclusive [Oryza meridionalis  and Oryza rufipogon . Oryza sativa was domesticated from Asian O. rufipogon 10,000 years ago [O. rufipogon chloroplast genomes are more similar to those of Australian O. meridionalis than to those of Asian O. rufipogon [Oryza AA genome species, a robust phylogenetic tree was established, which will aid in improving rice crops and in conservation strategies [There are several published chloroplast genomes from cereals, including those from sorghum  , wheat (estivum) , rye  , and ric sativa) . Rice isnclusive . For exaears ago . Neverthufipogon –67. Usinrategies , 5.Gossypium hirsutum) chloroplast genome was published in 2006 [Gossypium species comprise eight genome groups (A to G and K genomes). Gossypium hirsutum (upland cotton), the most widely planted cotton species in the world, is an allotetraploid of the ancestral A and D genome species [Gossypium species and these can be used to glean information about the evolution and domestication of this crop [Gossypium, including all eight genome groups and allotetraploid species [Gossypium herbaceum and Gossypium arboretum, were not cytoplasmic donors of tetraploid (AD) species; instead, the AD genome species originated from an extinct ancestor species of the modern A genome [Cotton is the most important textile fiber crop and the first cotton  chloroplast genome sequence [Phaseolus and Vigna chloroplast genomes [Information on chloroplast genomes is useful for understanding the domestication of several crops, particularly legumes . The chlsequence  before bsequence , 73–77.  genomes , 79. Mor genomes  and 5.6- genomes  inversio genomes  but alsoCitrus is one of the most commercially important fruit genera. In 2006, the first Citrus chloroplast genome, that of sweet orange (Citrus × sinensis), was published [Citrus (28) and Citrus-related genera (6) indicated that citrus fruits have the same common ancestor [matK, ndhF, ycf1, and ccsA), single-nucleotide variations and insertion/deletion frequencies were clearly higher than average and showed that these genes have been positively selected. The matK gene encodes a maturase that is involved in splicing type II introns and the matK sequence is often used in phylogenetic and evolutionary studies [matK is observed not only in citrus but is common in several other plant species. In fact, more than 30 plant groups have been shown to undergo positive selection of matK genes, indicating that the gene is subject to a number of different ecological selective pressures [ndhF gene encodes a subunit of the chloroplast NAD(P)H dehydrogenase (NDH) complex. Chloroplast NDH monomers are sensitive to high light stress, suggesting that the ndh genes may also be involved in stress acclimation [matK and ndhF show positive selection in Australian species, potentially contributing to their adaptation to a hot, dry climate [ublished  and thisublished , 84. Phyancestor . In fourressures . The ndhlimation . These s climate .Bamboo is an economically and ecologically important forest plant in Asia . Bamboo There are three distinct genomes in plant cells: nuclear, mitochondrial, and plastid. Mitochondria are believed to have evolved from a single endosymbiotic event by the uptake of a proteobacterium, whereas chloroplasts evolved from endosymbiosis of a cyanobacterium, after which there was a massive transfer of genes from the chloroplast to the nucleus . There ainfA, rpl22, and ndh from the chloroplast genome and their intracellular transfer to the nuclear or mitochondrial genomes provide valuable information for phylogenetic analyses and evolutionary studies. It is very easy to identify the chloroplast origin of genes in plant mitochondrial or nuclear genomes [Gene content, number, and structure are conserved in the chloroplast genome sequences of most autotrophic land plants , 98 but  genomes , 100 by  genomes , but thitranslation initiation factor 1 (infA) is a homolog of the essential gene infA in Escherichia coli [infA have occurred during angiosperm evolution [infA genes have been identified in Arabidopsis thaliana, soybean, tomato (Solanum lycopersicum), and ice plant  [infA in these four species contain chloroplast transit peptides. Studies using soybean and A. thaliana infA-GFP proteins have shown that nuclear-encoded infA genes are translated in the cytosol and transported into chloroplasts [infA deletions have been identified recently  ) and potential escape of transgenes via pollen.Engineering the introduction of foreign genes into the chloroplast genome addresses both of these concerns. Just two copies of transgenes are typically introduced into the nuclear genome, whereas up to 10,000 transgene copies have been engineered into the chloroplast genome of each plant cell, resulting in extremely high levels of foreign gene expression (>70 % TSP) . Most imThe basic process of chloroplast engineering is explained in Fig. aadA gene, which confers resistance to streptomycin and spectinomycin. These antibiotics bind specifically to chloroplast ribosomes and disrupt protein synthesis without interfering with any other cellular process. Efforts to transform the chloroplast genome of cereal crops have been mostly unsuccessful. This could be due to the instability of chloroplast DNA in the mature leaves of cereals [One of the challenges of creating chloroplast transgenic (transplastomic) plants is the elimination of all untransformed copies  of the native chloroplast genome and replacing them with transformed genomes that contain integrated transgene cassettes. The absence of the native chloroplast genome and the presence of only the modified genomes is referred to as the homoplasmic state, which is typically achieved after two or three rounds of selection Fig. . The mos cereals  or to a  cereals .Table Figure The selection of a suitable intergenic spacer region from among more than 100 sites found in each chloroplast genome is a major concern. Statements on the lack of positional effects in the transplastomic literature are common and are used to contrast chloroplast genetic engineering with nuclear transgene integration, which is often associated with profound differences in the expression of transgenes dependent on their site of integration. Evidence shows, however, that there are also positional effects within the chloroplast genome Table . IR regirbcL/accD) are located on opposite strands of the chloroplast genome, or transcriptionally active spacer regions, where native genes  are located within operons on the same strand. After a herbicide resistance gene was introduced into the transcriptionally active spacer region for the first time [trnI/trnA genes (used as flanking sequences) also provide efficient processing of native or foreign transcripts. The trnA gene intron includes a chloroplast origin of replication and produces more copies of the template (chloroplast vectors) for integration of the transgene cassette [trnA/trnI site of the chloroplast genome, confirming the unique advantages of this site [An early controversy in the chloroplast genetic engineering field was the suitability of transcriptionally silent spacer regions, where native genes  and downstream (3′ UTR) of transgenes play a major role in determining their expression level. The ears ago , still aoroplast  and it coroplast . Indeed,psbA regulatory region was used in tobacco chloroplasts or vice versa, transgene expression was dramatically reduced  resulted in changes that decreased the interaction of RNA-binding proteins and produced variation in the size of the stem, bulge, and terminal loop of the UTR [Using regulatory regions from photosynthetic genes has the advantage of light regulation, making them ideal for transgene expression in photosynthetic organs  was first evaluated for expression in leaves [BETAINE ALDEHYDE DEHYDROGENASE (BADH) was regulated by the T7 gene10 UTR in carrot (Daucus carota) plants, 75 % of the expression level in leaves was observed in non-green edible roots, conferring the highest level of salt tolerance (400 mM NaCl) found in the published literature  operon [Helicoverpa sp.) were killed with a single bite of leaf and insects that had 40,000-fold increased resistance to Bt were also killed  operon . Use of ) operon . These hled Fig. . NeverthErwinia soft rot and tobacco mosaic virus (TMV), which result in yield loss in several cultivated crops [Pinellia ternata agglutinin (PTA) gene in the chloroplast genome [Brassica oleracea) [Solanum melongena) [In the past decade, chloroplast genetic engineering has focused primarily on the overexpression of target genes with the potential to enhance biotic stress tolerance, which is very important for plant protection and yield enhancement. Yield loss due to insect pests can be very serious in many countries. In addition to cotton bollworm resistance conferred by hyper-expression of Bt protein in chloroplasts , there aed crops . Whitefled crops , which rt genome . More thleracea) , soybeanleracea) , 145, anlongena) .Helicoverpa insects decreased to almost undetectable levels in the midgut, resulting in a significant reduction in the net weight of larvae and in pupation rate [β-actin-targeting long dsRNA were lethal to Colorado potato beetle (Leptinotarsa decemlineata) larvae, providing yet another crop protection mechanism [More recently, scientists have begun to explore new strategies to downregulate specific target genes. One such approach is to express double-stranded RNAs (dsRNAs) within the chloroplast genome and to use RNA interference (RNAi) to confer the desired agronomic traits, mainly resistance to insects that cause severe yield loss. This strategy has been demonstrated by expressing long or short dsRNAs that activate RNAi and disrupt target genes in insects, providing efficient protection against insects without the need for chemical pesticides. One such example is the suppression of three essential proteins required for insect survival—lepidopteran chitin synthase (Chi), cytochrome P450 monooxygenase (P450), and V-ATPase—using dsRNAs in the tobacco chloroplast system . Each dsion rate . Transplechanism .E. coli or tobacco chloroplasts using a new technique that enabled the insertion of fungal genes with several introns, eliminating the need to prepare cDNA libraries. Industrial fermentation systems are currently limited by high cost and low production capacity; chloroplast-derived enzyme cocktails offer several striking advantages, including significantly reduced cost, improved stability of chloroplast-derived enzymes, and no need for enzyme purification. Interestingly, expression of β-glucosidase released hormones from conjugates, resulting in elevated phytohormone levels and increased biomass [In addition to improved resistance against both biotic and abiotic stress, the chloroplast genome has been engineered to produce useful enzymes, biomaterials, and biofuels, or even to enhance biomass. The first report of metabolic engineering using chloroplast genomes produced the highest level of the poly(p-hydroxybenzoic acid (pHBA) polymer (25 % dry weight) in normal healthy plants despite the diversion of a major metabolic intermediate . The fir biomass , an unexBrassica napus), and maize, are the major dietary source of vitamin E. They have very low α-tocopherol content but relatively high levels of γ-tocopherol. Only a few seed oils, such as sunflower (Helianthus annuus) seed oil, contain high levels of α-tocopherol, an important precursor of vitamin E [γ-tmt gene into the chloroplast genome resulted in the formation of multiple layers of the inner chloroplast envelope , with obvious phenotypic changes , pOral delivery of several human therapeutic proteins expressed in chloroplasts is highly efficacious in the treatment of several human diseases, including diabetes, cardiovascular disease, pulmonary hypertension, and Alzheimer’s disease. Most proteins were expressed in tobacco chloroplasts for initial evaluation and were subsequently expressed in lettuce chloroplasts for advancing them to the clinic. Oral delivery of exendin-4, which modulates the secretion of insulin in a glucose-dependent manner, lowered glucose in diabetic animals by stimulating the production of insulin in a manner similar to that of the injectable drug . Oral de2 hydroponic cGMP facility, it is possible to produce up to 30,000 doses for a 20-kg pediatric patient. Clotting factor made in lettuce was stable for up to 2 years when lyophilized cells were stored at ambient temperature, completely eliminating the need for the cold chain. This enables the first commercial development of an oral drug and addresses the extremely expensive purification, cold storage and transportation, and short shelf-life of current protein drugs. Oral delivery of a broad dose range was effective in the prevention of antibody formation after injection of clotting factor IX (FIX), further facilitating human clinical studies.The first industrial-scale production of human blood clotting factor in a cGMP facility was reported recently  Fig. 4a. In a 10The current iteration of vaccines, using attenuated bacteria or viruses, offer protection against major infectious diseases but they also present major challenges. For example, the oral polio vaccine that is used around the globe has caused severe polio resulting from mutations and recombination with other viruses . In addiVibrio cholerae was fused to the malarial vaccine antigen apical membrane antigen-1 (AMA1) and merozoite surface protein-1 (MSP1) and expressed in lettuce or tobacco chloroplasts. While no suitable models exist to test human malaria, a cholera toxin challenge using mice immunized with chloroplast-expressed CTB was highly effective and provided the longest duration of protection in the published literature [One successful chloroplast-derived vaccine conferred dual immunity against cholera and malaria in animal studies . Choleraterature . These eterature  for whicBt corn [It is amazing that the chloroplast genome can express >120 foreign genes from different organisms, including bacteria, viruses, fungi, animals, and humans. The insertion of commercially useful traits, including herbicide and insect resistance, into soybean resulted in high-level expression and superior transgene containment, with no antibiotic selectable markers; but even so, these lines were not developed commercially. Nevertheless, recurring concerns about insect resistance against biopesticides have resulted in new USDA requirements on planting Bt corn , which mAlthough products with high-level expression have now advanced to the clinic or are in commercial development, a better understanding of chloroplast translation is required to improve several other gene products. The availability of chloroplast genome sequences should help in the development of codon optimization programs using highly expressed chloroplast genes, but among the ~3000 cultivated crops, sequenced chloroplast genomes are available for crops from fewer than 70 genera. Major funding agencies have not supported crop chloroplast genome sequence projects because of the misconception that all chloroplast genomes are similar, as evidenced by the publication of fewer than ten crop chloroplast genome sequences between 1986 and 2004. This review illustrates the importance of sequencing more crop chloroplast genomes for various biotechnology applications. Furthermore, new selectable markers are needed to transform the chloroplast genomes of cereals, which has been elusive for the past two decades.Chloroplast genome sequences will be valuable assets in herbal medicine. Most medicinal plants are rare species and very little information is available to confirm their identity. DNA barcodes derived from chloroplast genomes will be useful for identifying varieties and resources; this concept is also valuable in the identification of the origin of cultivated crops and their close relatives to enhance breeding or transfer of useful traits. Molecular techniques to sequence the genomes of single chloroplasts could help to eliminate chloroplast-like sequences that are present in the mitochondrial or nuclear genome. The ability to sequence chloroplast genomes using minimal leaf materials could help us to understand variations in different segments of a variegated leaf in horticultural crops. Further, determining complete chloroplast genome sequences from fossils or recently extinct plants could shed more light on chloroplast genome evolution; help us to understand these species’ inadequate fitness to cope with environmental changes; and help us to build new phylogenetic trees. The technology for isolating DNA from fossils is already available –168. AllBt, Bacillus thuringiensis; CTB, Cholera toxin B subunit; cGMP, Current Good Manufacturing Processes; CTB, Cholera toxin B subunit; dsRNA, double-stranded RNA; FIX, clotting factor IX; infA, translation initiation factor 1; IR, inverted repeat; LSC, large single copy; ndh, NAD(P)H dehydrogenase; NCBI, National Center for Biotechnology Information; NGS, next-generation sequencing; RNAi, RNA interference; SSC, small single copy; γ-TMT, γ-Tocopherol methyltransferase; TSP, total soluble protein; UTR, untranslated region; Ang (1–7), Angiotensin (1–7)ACE2, Angiotensin-converting enzyme 2; BAC, bacterial artificial chromosome;


Current opinions with respect to the etiology of chronic diseases such as neurodegenerative disease, cardiovascular diseases, and cancer are still controversial. There are multiple factors involved, and oxidative stress that results from the imbalance between reactive oxygen species and antioxidants is a one of the key factors in the development of these chronic diseases. Indeed, experimental and clinical evidences support the casual relationship between oxidative stress and various chronic diseases. Thus, numerous studies are focused on ameliorating these chronic diseases by reducing the oxidative stress. Epidemiological and clinical studies suggest that natural products can combat oxidative stress and reduce the morbidity and mortality associated with chronic diseases. Many natural compounds such as flavonoids are potential antioxidants that protect against reactive oxygen species (ROS) or reactive nitrogen species- (RNS-) induced damage and ameliorate oxidative stress-related diseases, such as neurodegenerative diseases, cardiovascular diseases, inflammatory conditions, and cancer. Although various natural products have been shown to possess potential protective effects against chronic diseases, the bioactivities of large number of natural compounds remain unknown. Therefore, understanding and validating the bioactivities of the natural compounds and the molecular mechanisms involved would provide solid scientific foundation to use the natural compounds for the prevention and treatment of oxidative stress-related diseases. The primary objective of this issue is to highlight the role of some of the natural products that ameliorate oxidative stress and reduce pathological complications associated with chronic diseases. The articles published in this special issue include four reviews and four original research articles that describe the importance of some of the natural products.The review by J. Pérez-Hernández et al. is focused on the possible role of antioxidant herbal compounds as an alternative source for the treatment of neurodegenerative diseases (ND). The herbal compounds discussed in this review include polyphenols, flavonoids, alkaloids, and other miscellaneous antioxidant compounds from plants. An interesting review article by Y. Bai et al. discusses the cardiovascular beneficial effects of sulforaphane, a sulfur-based isothiocyanate compound in the cruciferous vegetables. Clinical and animal studies showed sulforaphane improved cardiovascular complications through activation of Nrf2. In this review, the authors discuss the possible molecular mechanisms involved in sulforaphane induced Nrf2 activation. J. A. Sirerol et al.'s review is focused on the role of natural compound stilbene in the prevention of cancer. The authors discuss about the studies that support the anticancer effects of stilbene, which also include few clinical trials. In this issue, the review article by J. Wang et al. discusses the latest advancements and up-to-date discoveries on the mechanisms of antioxidant activity of polysaccharides and glycoconjugates derived from natural products.α-lipoic acid (ALA) and superoxide dismutase (SOD) can counteract senile neurodegenerative deterioration to the retina and optic nerve. This study indicates that the combination of ALA and SOD could reduce oxidative stress and thereby prevent nuclear degradation and the subsequent cell death.ROS play a major role in the aging process and significantly contribute to the aging related pathological complications. The antioxidant system is compromised with aging and the antioxidant levels are no longer sufficient to counteract the generation of free radicals. Exogenous antioxidants may be a promising strategy to suppress oxidative stress associated with aging. In this issue, the original research article by M. Nebbioso et al. shows that  Aronia melanocarpa concentrate attenuated paraquat-induced neurotoxicity. In this study, Aronia berry concentrate at low doses can restore the homeostatic redox environment of neurons treated with paraquat, while high doses exacerbate the imbalance leading to further cell death. This suggests that moderate levels of Aronia berry concentrate may prevent reactive oxygen species-mediated neurotoxicity whereas high doses may have toxic effects. The study by H. Lv et al. shows that Licochalcone A (Lico A) enhances Nrf2-mediated defense mechanisms against tert-butyl hydroperoxide (t-BHP) induced oxidative stress and cell death via Akt and ERK activation in RAW 264.7 cells. Further, their results indicate that Lico A might modulate HO-1 and scavenge ROS via the activation of the PI3K/Akt and ERK/Nrf2 signaling pathways.In everyday life, humans are readily exposed to toxicants derived from various sources such as food , pesticides , and industry . These toxic substances trigger oxidative stress in the biological systems, which consequently lead to acute or chronic diseases. In this issue, W. Chen et al. reported that wild raspberry can improve the protective capacity against ethyl carbamate-induced oxidative damage in Caco-2 cells. Their results demonstrate that raspberry extract subjected to simulated gastrointestinal digestion could improve the cellular antioxidant activity and attenuate ethyl carbamate-induced oxidative damage in Caco-2 cells. A. J. Case et al.'s research article shows thatIn summary, this special issue covers a series of topics addressing the role of natural products as antioxidants counteracting oxidative stress-related chronic diseases. We hope that the articles brought in this special issue not only enrich our understanding of the therapeutic role of natural products on oxidative stress-related disease but also provide promising perspectives on future novel therapeutic agents development.Wei ChenWei ChenZhenquan JiaZhenquan JiaMin-Hsiung PanMin-Hsiung PanPon Velayutham Anandh BabuPon Velayutham Anandh Babu


Angiogenesis is essential for the growth and metastasis of solid tumors. The tumor endothelium exists in a state of chronic activation and proliferation, fueled by the tumor milieu where angiogenic mediators are aberrantly over-expressed. Uncontrolled tumor growth, immune evasion, and therapeutic resistance are all driven by the dysregulated and constitutive angiogenesis occurring in the vasculature. Accordingly, great efforts have been dedicated toward identifying molecular signatures of this pathological angiogenesis in order to devise selective tumor endothelium targeting therapies while minimizing potential autoimmunity against physiologically normal endothelium. Vaccination with angiogenic antigens to generate cellular and/or humoral immunity against the tumor endothelium has proven to be a promising strategy for inhibiting or normalizing tumor angiogenesis and reducing cancer growth. Here we review tumor endothelium vaccines developed to date including active immunization strategies using specific tumor endothelium-associated antigens and whole endothelial cell-based vaccines designed to elicit immune responses against diverse target antigens. Among the novel therapeutic options, we describe a placenta-derived endothelial cell vaccine, ValloVax™, a polyvalent vaccine that is antigenically similar to proliferating tumor endothelium and is supported by pre-clinical studies to be safe and efficacious against several tumor types. Angiogenesis, the outgrowth of new blood vessels from pre-existing capillaries and post-capillary venules, occurs during embryonic development, in the uterus during the menstrual cycle, in the process of wound healing, and in pathological conditions . In healThe goal in vaccination strategies is to raise immunity against antigens present in tumor endothelium while avoiding antigens that cross-react with healthy vasculature, thereby preventing deleterious autoimmune reactions. Since the landmark publication by Dr. Folkman, a catalog of molecular players involved in the process of tumor angiogenesis have been identified and characterized. However, clinical outcomes of traditional anti-angiogenic therapies such as monoclonal antibodies have improved patient survival rates only modestly . VaccinaEndothelium is a dynamic and heterogeneous structure influenced by environmental factors such as shear stress, oxygen content of the blood, chemokines, cytokines, and changes in the content of the extracellular matrix . WhereasMDR1, a gene encoding a transmembrane glycoprotein P-gp that is known as a multidrug transporter that potentiates resistance to several anti-cancer agents [Although tumor endothelial cells are considered to be genetically stable as compared to tumor cells, angiogenic factors can fuel genetic aberrations of tumor endothelium. Indeed, resistance of tumor endothelial cells in diverse cancers has been described; for example, tumor endothelial cells in renal carcinoma are reportedly resistant to serum starvation , breast r agents . For exar agents . VaccinaHeterogeneity among tumor endothelial cells, as choreographed by the over-activity of certain angiogenic pathways, also shields tumors by affecting their visibility to the immune system. Production of angiogenic molecules by tumors inhibits the expression of adhesion molecules involved in leukocyte interactions with blood vessel walls, including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin and CD34 . These fCancer vaccines consist of tumor-associated antigens delivered in a pro-inflammatory context to generate potent antitumor immune responses and overcome the cancer’s varied immunosuppressive mechanisms. Vaccination strategies directed against tumor-associated endothelium are designed to take advantage of both quantitative and qualitative differences between tumor endothelial cells and non-malignant endothelial cells. Ideal vaccine candidates include receptors/proteins that are upregulated in tumor endothelium, owing to its activated and proliferating state, but are sparsely expressed during physiological angiogenesis in healthy adult tissues. Notably, angiogenesis-associated molecules can be over-expressed on endothelial cells in an organ- or tumor-specific manner, and can sometimes be expressed on resting endothelial cells . Thus, aWhether immunity and/or autoimmunity ensue as a result of vaccination is dependent not only on the target tumor endothelial antigen but also on the vaccination protocol. Candidate vaccines have employed numerous delivery approaches, including proteins, peptides, dendritic cells pulsed with the antigen(s), naked DNA or recombinant DNA delivered by carriers, and mRNA vaccines, as well as whole cell vaccines or endothelial membrane components. To develop rationales for clinical vaccine design, pre-clinical studies have been conducted for many angiogenesis-associated molecules using various delivery systems. In some cases, differences in safety and efficacy between vaccines hinge on a plethora of secondary variables including the use of different delivery vectors, the choice of adjuvants and the varying routes of vaccine administration.Two general approaches to anti-angiogenesis vaccination have yielded promising results for inducing specific and robust immunity against tumor endothelium and reducing tumor growth and metastasis: (1) vaccines expressing defined angiogenesis-associated antigens; and, (2) vaccines comprised of whole endothelial cells and/or mixtures of endothelial antigens. In the latter category are vaccines consisting of placenta-derived endothelial antigens, an approach currently being advanced by Batu Biologics, Inc. .Evidence has shown the feasibility of targeting molecules that are expressed by angiogenic endothelium, and the data also suggest that anti-angiogenic vaccination can be applied synergistically with tumor immunotherapy and/or chemotherapy to invoke anti-tumor immunity. Theoretically, the approach of targeting certain defined angiogenesis-associated molecules on tumor endothelium has the risk to evoke only a transient decrease in cancer progression, owing to the fact that angiogenesis can proceed via compensatory pathways. Despite this caveat, success at achieving anti-angiogenic immunity has been demonstrated by vaccinating against the following antigens.Xenopus VEGF DNA vaccine that was protective and therapeutic in several tumor models in mice, an effect that was mediated by anti-tumor activity of CD4+ lymphocytes [VEGF/VEGFR is perhaps the best-studied angiogenic pathway involved in the growth and survival of tumor endothelium. Clinical therapies addressing tumor angiogenesis have therefore largely focused on inhibiting VEGF and its cognate receptor. VEGF-A exists in several pro-angiogenic variants that are secreted by both tumor cells and endothelial cells and activate VEGFR1 and VEGFR2. Numerous VEGF-expressing vectors have been administered for vaccinating against VEGF. A phocytes . A phasephocytes . After ephocytes –30, no aVEGFR2 , which is highly expressed on proliferating endothelial cells of the tumor vasculature, has also been the focus of numerous pre-clinical studies. Animal models using VEGFR2 DNA, protein, and peptide vaccines have demonstrated their ability to elicit potent humoral and cellular immunity, suppression of angiogenesis, tumor necrosis and/or suppression of metastastatic progression –38. InteSince VEGFR2 is also expressed at lower levels in normal vascular endothelium, vaccination could theoretically cause side effects or an autoimmune response. Neithammer et al. reported that in murine models a DNA vaccine against VEGFR2 suppressed angiogenesis in the tumor vasculature as verified by in vitro inhibition of endothelial cell proliferation, deposition of antibodies in tumor vasculature, a reduction of microvessel density and antitumor activity in vivo without impairment of fertility, neuromuscular performance or hematopoiesis; however, a slight delay in wound healing in immunized mice was noted . In anotImmunogenic epitopes of VEGFR2 have been identified and peptide vaccines have been optimized for binding to MHC class I molecules in order to elicit activity of cytotoxic T lymphocytes (CTL) specific for tumor endothelium. In a clinical study of pancreatic cancer, administration of immunogenic VEGFR2-169 peptide vaccine in combination with gemcitabine was well tolerated with no severe adverse events and peptide-specific CTL were induced . Other tBasic fibroblast growth factor (bFGF or FGF-2) and its receptor  have been targeted in pre-clinical mouse models to evaluate their anti-angiogenic and anti-tumor effects. FGFR-1 is highly expressed on angiogenic endothelium as well as on endothelial progenitor cells , 47. In The expression of several cell adhesion molecules, most notable integrin alpha(v)beta(3), has been associated with tumor angiogenesis .Integrin αvβ3 plays a role in several physiological processes including angiogenesis, pathological neovascularization, and tumor metastasis . An αvβ3Another target of anti-angiogenic vaccine strategies is the angiostain binding protein angiomotin, a membrane-associated pro-angiogenic protein present on endothelial cells in angiogenic tissues . HolmgreThis transmembrane glycoprotein is primarily expressed in proliferating endothelial cells and is upregulated by hypoxia; therefore, endoglin is strongly expressed in tumor endothelial cells . The funThis is an intracellular tumor-associated antigen that is upregulated in cancers and tumor endothelial cells but is not expressed in healthy differentiated tissues. Survivin is a member of the inhibitor of apoptosis protein family, and serves to inhibit programmed cell death, promote cellular proliferation and enhance angiogenesis . SurviviRobo4 is a member of the Roundabout family proteins and is a transmembrane cell adhesion molecule that is specifically expressed in endothelial cells and hematopoietic stem cells and progenitor cells . Robo4 gTie-2 is an endothelium-specific receptor tyrosine kinase that binds to angiopoietin and is upregulated on diseased endothelium in diverse conditions including cancer, atherosclerosis, and macular degeneration. A protein vaccine based on chicken Tie-2 reduced growth of hepatomas and melanomas in mice, whereas a murine Tie-2 vaccine was ineffective, indicating the need for further enhancement of this protein’s immunogenicity . The chi®; and a EGFR-based HER-1 cancer vaccine. CIMAvax-EGF is a therapeutic cancer vaccine of human recombinant epidermal growth factor (EGF) conjugated to a carrier protein from Neisseria meningitides. The vaccine induces antibodies against EGF and confers a survival advantage in patients with non-small-cell lung cancer [This receptor can be activated by diverse ligands; namely, EGF, transforming growth factor-α (TGF-α), amphiregulin, betacellulin, heparin-binding EGF, or epiregulin that are locally secreted by cancer cells and can act in an autocrine fashion. EGFR signaling regulates the secretion of several different angiogenic growth factors by tumor cells including VEGF . Co-exprg cancer . Studiesg cancer  Fig. 1)®; and a 2 terminal that has no homology with any known protein; therefore, this antigen could be used for raising specific immunity against the pathological vasculature in human cancer [This polypeptide is expressed as a marker for neonatal lung vasculature and adult pathological angiogenesis. HP59 protein has been identified in the vasculature of many types of tumors including lung, colon, ovary, and breast cancers of different stages, but not in the corresponding normal tissues; therefore, this protein could be an ideal target antigen for vaccinating against tumor endothelium . Vaccinan cancer .This tyrosine kinase receptor is activated by members of the platelet-derived growth factor family, and is broadly involved in regulating cellular proliferation and differentiation in physiological and pathological contexts. PDGF-B, the ligand, produced by tumor cells binds to PDGFR on stromal and perivascular cells to promote tumor growth and angiogenesis, as well as eliciting other pathways of tumor nourishment , 84. PDGAnti-PDGF drugs have been widely used for treating various cancers; however, their mechanisms of action at a molecular level are not strictly anti-angiogeneic. Paradoxically, PDGF ligand inhibition can increase tumor angiogenesis, which supports increased tumor growth but, in doing so, normalizes blood vessel walls to allow for more efficient delivery of chemotherapy drugs , 86. On Salmonella typhimurium was given to mice orally in murine colon, breast and lung carcinoma model systems [To assess the effects of vaccinating against PDGFRβ, a DNA vaccine delivered via  systems . SignifiTem1 cDNA fused to tetanus toxoid adjuvant inhibited tumor growth and progression without affecting reproduction or wound healing [Tumor endothelial marker 1, also known as endosialin or CD248, is a type 1 membrane protein involved in developmental, physiological and pathological angiogenesis. TEM1 is overexpressed in the vasculature of carcinomas, brain tumors and sarcomas –91 and i healing . SplenocSalmonella typhimurium that was capable of generating TEM8-specific CD8+ cytotoxic T cells and protected mice from lethal tumor challenge [Designing a tumor endothelial vaccine based on TEM8 is a very enticing possibility since this molecule is selectively upregulated on tumor endothelial cells and, unlike the majority of other putative vaccine targets, TEM8 is undetectable in endothelium during physiological angiogenesis . The firhallenge . Anotherhallenge . Lastly,hallenge . This vaIn summary, several therapeutic approaches have been explored targeting single antigen expressed on the tumor endothelium, demonstrating the feasibility of targeting the tumor vasculature by vaccine therapy without inducing systemic autoimmunity to quiescent endothelium in vivo. Signals of efficacy have been observed in preclinical studies around these targets, and several tumor endothelium-targeting vaccines have made it into the clinical setting for the treatment of cancer. As such, in an effort to reduce the development of treatment resistance and to potentiate the strength of an immune response and resultant survival benefit, the authors would like to explore the concept of a polyvalent vaccine targeting several angiogenesis associated antigens.Although great strides have been made in advancing anti-angiogenesis tumor vaccines against specific targets, it is clear that the presence of many interrelated and compensatory pathways, as well as genetic instability of tumor endothelium could theoretically overcome targeted inhibition in a clinical setting. Accordingly, polyvalent vaccination approaches are being developed using whole endothelial cells or isolated proteins from endothelial cell membranes. Another advantage of such polyvalent vaccines expressing numerous angiogenic antigens is to allow antigen-presenting cells to process and present immunodominant epitopes for generating anti-angiogenic immunity.Human umbilical vein endothelial cells (HUVEC) have been the standard for cell-based models of tumor angiogenesis, having the ability to proliferate extensively and expressing a number of pro-angiogenic molecules that mimic tumor neovasculature. Specifically, the aforementioned antigens such as VEGFR2, αvβ3 and endoglin, which are common biomarkers associated with tumor angiogenesis, are expressed in the primary culture of HUVECs . In a reHUVECs have been used in pilot studies to test the anti-angiogenic effects of vaccination in patients with malignant brain tumors and metastatic colorectal cancer , 107. VaIn a related study, 17 patients with recurrent glioblastoma were treated with the HUVEC vaccine using the same protocol of intradermal delivery . These pAmong the vaccine candidates for targeting tumor endothelium, the placenta is a source of significant numbers of proliferating and angiogenic cells owing to its immunological status at the maternal-fetal interface. In the placenta, trophoblast cells invade into the maternal uterine wall where they remodel the spiral arteries and supply angiogenic factors for de novo blood vessel formation and expansion of the pre-existing vascular network . These pThe idea of using placenta as a source of antigens for vaccination against tumors originated in the 1970s from Dr. Valentin Govallo who noted the immunological similarities between pregnancy and cancer and demonstrated that immunity to placental trophoblast cells conferred radiological tumor reductions in lung cancer patients (reviewed in ). IndeedBatu Biologics is currently advancing ValloVax™, a vaccine consisting of placenta-derived endothelial cells pretreated with interferon gamma to enhance immunogenicity. Pre-clinical studies revealed that ValloVax™ potently inhibits tumor growth in 3 histologically distinct animal models and also suppresses pulmonary metastasis subsequent to intravenous tumor administration . This neBlocking tumor-induced angiogenesis has become a focal point for the development of new cancer therapeutic drugs. Vaccines represent a promising approach for overcoming tolerance to the angiogenic factors that support cancer growth and metastasis. Antigens that are highly expressed on proliferating tumor endothelium, while comparatively downregulated or absent on quiescent endothelium in healthy tissues and during normal physiological angiogenesis are ideal candidates for vaccination strategies targeting the tumor vasculature. Two major types of vaccines are being developed; namely, vaccines against defined angiogenesis-associated antigens, and whole endothelial cell vaccines. The data available thus far support the feasibility of these approaches for generating humoral and cell-mediated immunity against the tumor vasculature and the safety of endothelial cell-based vaccines.