Visual categorization may already start within the first 100-ms after stimulus onset, in contrast with the long-held view that during this early stage all complex stimuli are processed equally and that category-specific cortical activation occurs only at later stages. The neural basis of this proposed early stage of high-level analysis is however poorly understood. To address this question we used magnetoencephalography and anatomically-constrained distributed source modeling to monitor brain activity with millisecond-resolution while subjects performed an orientation task on the upright and upside-down presented images of three different stimulus categories: faces, houses and bodies. Significant inversion effects were found for all three stimulus categories between 70–100-ms after picture onset with a highly category-specific cortical distribution. Differential responses between upright and inverted faces were found in well-established face-selective areas of the inferior occipital cortex and right fusiform gyrus. In addition, early category-specific inversion effects were found well beyond visual areas. Our results provide the first direct evidence that category-specific processing in high-level category-sensitive cortical areas already takes place within the first 100-ms of visual processing, significantly earlier than previously thought, and suggests the existence of fast category-specific neocortical routes in the human brain. We have the remarkable ability to recognize thousands of visual objects in our daily environment, such as faces, bodies, cars, keys, shoes, animals, food, tools, and houses. Despite its complexity, visual categorization is executed rapidly and effortlessly by the human brain. These computations appear to be mainly carried out by the ventral visual pathway Functional neuroimaging studies ) in humans have examined the higher-level cortical regions involved in the visual perception of different objects. Faces For instance, intracranial recordings in patients have shown that during face perception the well-established face-selective area of the fusiform gyrus becomes strongly activated around 170-ms after stimulus onset However, this traditional model of object perception is challenged by recent psychophysical and electrophysiological findings suggesting that visual categorization processes may already take place at even earlier latencies The neuronal underpinnings of this proposed early phase of visual categorization analysis remain however a puzzle. Reports on the neuronal origin of the P1/M100 response to faces have been inconsistent, as sources have been found in the retinotopic cortex of the medial occipital cortex We took advantage of the high temporal resolution of magnetoencephalography (MEG) combined with its relatively good spatial resolution to investigate whether category-specific cortical activation may already take place within the first 100-ms. Early visual electrophysiological responses are known to be extremely sensitive to the physical attributes of the stimulus For the present experiment, we selected faces as a stimulus category because they show a strong behavioral Bodies were chosen as a second type of biologically salient stimuli with strong configurational properties and a special perceptual status somewhat similar to that of faces Participants viewed photographs of faces, bodies and houses presented in either their upright or inverted orientation or in a Fourier phase-scrambled version, and were asked to classify them accordingly, i.e. as upright, inverted or scrambled  for the magnetometers and gradiometers and the mean global dipole strength over the cortex . When lot-statistics for the contrasts between the Upright and Inverted conditions  there was relatively little cortical overlap between categories . QuantitExtraction of the time-courses of the estimated currents from the six main regions that showed a significant Inversion Effect  confirmeWe investigated whether category-specific cortical activation in the human brain already takes place within the first 100-ms of visual processing by comparing the MEG inversion effects of three stimulus categories: faces, bodies, and houses. We found that the first prominent MEG component peaking around 100-ms after stimulus onset (M100) was already sensitive to stimulus inversion of all three investigated object classes . SignifiImportantly, category-specific cortical activation was identified on the basis of intra-categorical comparisons, i.e., we compared the responses to the upright stimuli with the responses to the same images when presented upside-down. As such we attempted to avoid confounds associated with the low-level physical properties of the stimuli. A nonspecific general inversion effect was found in the retinotopic areas of the medial occipital lobe (∼V1/V2), and an overlap between the face and house inversion effect was encountered in the right lateral occipital cortex. Although we cannot rule out that the effects in retinotopic areas may partly be caused by systematic low-level differences between the upper and bottom half of the stimuli, three important findings count in favor of category-specific processing. First, category-specific inversion effects were found well beyond retinotopic areas, even in prefrontal areas such as the posterior IFG and the medial orbitofrontal area. Second, the areas sensitive to stimulus inversion showed a strong hemispheric lateralization. Third, the cortical distribution of the face inversion effect was exactly found in those areas that are known to exhibit face-selective responses and that are part of the well-established distributed network for face processing e.g. : the infThe current result of an early inversion effect is consistent with previous MEG findings of an inversion effect for the M100–120 component The currently observed location of the M100 inversion effect which indicates the early activation of a distributed network, is however in contrast with previous source analysis studies of the M100–120 face inversion effect The general inversion effect found in the medial occipital cortex may be interpreted as the consequence of systematic differences in low-level properties  between the upper and bottom halves of the image and the reported asymmetry for lower half-field and upper half-field VEF responses The early MEG component in the fusiform gyrus corresponds to the initial potential around 90–110-ms recorded with intracerebral electrodes from the fusiform gyrus in human epilepsy patients Interestingly, we observed a small early body inversion effect. So far only one EEG study investigated the effect of body inversion on the P1 component The main location of the early differential activity for houses in present study, the right lateral occipital area, is in agreement with the hemodynamic inversion effect for scenes/houses Present findings of early category-specific activation of category-sensitive distributed cortical networks between 70–100-ms after stimulus onset are consistent with a growing body of evidence that visual categorization can already take place within the first 100-ms post stimulus onset The current results provide evidence for the existence of a rapid neocortical route in humans in which information is rapidly transferred from low-level visual areas to high-level category-sensitive visual areas. Our findings suggest that activation started focally in the medial occipital lobe around 50–60-ms, and from here propagated rapidly to more anterior, inferior and lateral areas, extending to the full ventral aspect of the temporal lobe within 100-ms post stimulus onset . The latDirect neurophysiological evidence for the neural mechanisms underlying object categorization in the visual ventral stream stems from monkey recordings, in which face-specific neuronal populations in high-level visual cortex have been found to become activated in two phases Our present MEG findings suggest that similar processes of a first and second pass through the ventral object system may also underlie object recognition in the human brain. The reasons why this first pass has not been fully recognized before may be that during the initial wave of activation sources in low-level occipital areas are far stronger than those in high-level areas, and that the activity in high-level cortical areas is far more prominent during the second wave of activation than during the first pass. To explore the neuronal mechanisms of this subtle first pass in more detail, it would be of interest to investigate whether the early category-specific activation is mediated by the fast magnocellular system which is sensitive to low-spatial frequencies and processes only the coarse information of the image. It has already been shown that low-spatial frequencies only can account for the fast interpretation of natural scenes In conclusion, our results provide the first direct evidence of fast category-specific neocortical routes in the human brain, hereby challenging the long-held view that during the first 100-ms only the low-level features of the stimulus are being processed, and that category-specific cortical activation only occurs at later stages.Ten healthy right-handed individuals  with normal or corrected to normal vision volunteered to take part in the experiment. All procedures were approved by the Massachusetts General Hospital Institutional Review Board, and informed written consent was obtained from each participant. The study was performed in accordance with the Declaration of Helsinki.Face stimuli were gray-scale photographs from the Ekman and Friesen database Since lower half-field stimuli evoke larger visual evoked fields than upper half-field checkerboards The experiment was divided into four blocks each consisting of 288 trials. Within one block, all stimuli (8 exemplars * 9 stimulus categories) were presented 4 times in random order, summing up to a total of 128 trials for each stimulus condition. Half of the subjects started responding with their left hand, while the other half started with their right hand. At each new block the participants changed the button box to their other hand. To familiarize the subjects with the procedure and task demands the experiment was preceded by a short training session, which contained all stimulus categories.The experiment was conducted in a magnetically shielded, sound-attenuating room . Subjects were comfortably seated with the head leaning against the back of the helmet of the MEG dewar. The visual stimuli were presented with a LP350 Digital Light Processor projector  onto a back-projection screen placed 1.5 m in front of the subject. The size of the framed stimuli on the screen was 17×17 cm, subtending a visual angle of 6.5°. The trial designation is depicted in MEG data were acquired with a 306-channel Neuromag VectorView system , which combines the focal sensitivity of 204 first-order planar gradiometers with the widespread sensitivity of 102 magnetometers. Eye movements and blinks were monitored with vertical and horizontal electro-oculogram. The location of the head with respect to the sensors was determined using four head-position indicator coils attached to the scalp. A head-based MEG coordinate frame was established by locating fiduciary landmarks (nasion and preauricular points) with a Fastrak 3D digitizer . The data were digitized at 600 samples/second with an anti-aliasing low-pass filter set at 200 Hz.MEG signals were averaged across trials for each condition, time-locked to the onset of the stimulus. A 34-ms delay between the time the computer sent an image and the time it was projected onto the screen was measured with a photodiode and subsequently taken into account when reporting the timing of measured activity. A 200-ms pre-stimulus period served as baseline. Trials to which subjects made an incorrect response and those that contained eye blinks exceeding 150 µV in peak-to-peak amplitude or other artifacts were discarded from the average. The evoked responses were low-pass filtered at 40 Hz.MEG data were co-registered with structural high-resolution magnetic resonance images (MRI). A set of 3-D T1-weighted MR images using a 1.5 T system were acquired. The MRI and MEG coordinate systems were aligned by identifying the fiducial point locations in the MRIs. In addition several points were digitized from the head surface to allow confirmation and fine tuning of the initial alignment based on the fiducial landmarks.The geometry of the cortical mantle was extracted from the MRI data using the Freesurfer software t-tests for paired samples (α = 0.05) on the MGFP and the mean current strength across dipoles respectively at successive time points.MEG data was first quantified at the sensor level. The mean global field power (MGFP) was calculated separately for the magnetometers and the gradiometers by squaring the signal values and averaging them across sensors. Another global measure was obtained by averaging the time courses of the estimated currents across all dipoles (see next section) in each individual brain. Statistical group analysis was performed by two-tailed The source current distribution was estimated at each cortical location using the minimum-norm estimate (MNE) t-tests  were performed between the Upright and Inverted conditions for each dipole location and each time point. The results were thresholded for the baseline noise, i.e. significant t-values at the level of p<0.01 were visualized only if the currents exceeded a signal-to-noise ratio of 2.5 (dSPM>2.5) in at least one of the two stimulus conditions. Next, the source distributions of the category-specific M100 Inversion Effects were determined by taking the largest significant (supra-baseline-noise) positive or negative t-values at each dipole location occurring within the 70–100-ms time-window.Group movies were created by morphing the source estimates for each individual subject to the cortex of one representative subject, according to the method of Fischl et al. The amount of spatial overlap between the category-specific M100 Inversion Effects was quantified by calculating the number of dipoles that met the M100 Inversion Effect criteria described above for one, two or three of the categories.t-tests for paired samples (α = 0.01) were performed on the mean current strength across dipoles at successive time points.To investigate the time courses of the main regions that showed significant group Inversion Effects in the average brain in more detail, the corresponding anatomical regions were drawn in the reconstructed inflated cortical surface of each individual. The regions selected were the calcarine sulcus, Inferior Occipital Gyrus (IOG), Lateral Occipital Cortex (LOC), the middle part of the Fusiform Gyrus (mFG), the transition region of the posterior Inferior Frontal Gyrus  and middle part of the superior circular insular sulcus, and the posterio-dorsal medial parietal areas (Precuneus and the posterior Cingulate Gyrus). For the region of the calcarine sulcus, we excluded the anterior half of the calcarine fissure, representing peripheral visual field eccentricities that were not stimulated in our protocol, and selected the posterior half of the calcarine fissure including a small strip (ca. 2 mm) of the adjacent gyri. The fusiform gyrus (FG) was divided into three parts along its anteroposterior axis, resulting into an anterior, middle and posterior part. The LOC comprised the anatomical regions of the middle occipital gyrus and sulcus and the anterior occipital sulcus. The time courses of the estimated currents for each dipole within these selected regions were extracted and used for further analysis. Two-tailed


Monochorionic (MC) twins are at increased risk for perinatal mortality and serious morbidity due to the presence of placental vascular anastomoses. Cerebral injury can be secondary to haemodynamic and hematological disorders during pregnancy  or intrauterine co-twin death) or from postnatal injury associated with prematurity and low birth weight, common complications in twin pregnancies. We investigated neurodevelopmental outcome in MC and dichorionic (DC) twins at the age of two years.This was a prospective cohort study. Cerebral palsy (CP) was studied in 182 MC infants and 189 DC infants matched for weight and age at delivery, gender, ethnicity of the mother and study center. After losses to follow-up, 282 of the 366 infants without CP were available to be tested with the Griffiths Mental Developmental Scales at 22 months corrected age, all born between January 2005 and January 2006 in nine perinatal centers in The Netherlands. Due to phenotypic (un)alikeness in mono-or dizygosity, the principal investigator was not blinded to chorionic status; perinatal outcome, with exception of co-twin death, was not known to the examiner.Four out of 182 MC infants had CP (2.2%) - two of the four CP-cases were due to complications specific to MC twin pregnancies (TTTS and co-twin death) and the other two cases of CP were the result of cystic PVL after preterm birth - compared to one sibling of a DC twin  of unknown origin. Follow-up rate of neurodevelopmental outcome by Griffith's test was 76%. The majority of 2-year-old twins had normal developmental status. There were no significant differences between MC and DC twins. One MC infant (0.7%) had a developmental delay compared to 6 DC infants . Birth weight discordancy did not influence long-term outcome, though the smaller twin had slightly lower developmental scores than its larger co-twin.There were no significant differences in occurrence of cerebral palsy as well as neurodevelopmental outcome between MC and DC twins. Outcome of MC twins seems favourable in the absence of TTTS or co-twin death. Monochorionic (MC) twins are at increased risk for perinatal mortality and serious morbidity compared to dichorionic (DC) twins Although long-term outcome has been studied extensively in TTTS All monochorionic (MC) twin pregnancies delivered between January 2005 and January 2006 in nine perinatal centers in The Netherlands, all tertiary referral centers with neonatal intensive care units (NICUs), were recorded. During the study period, 111 MC twin pairs were born in these hospitals. Eleven twin pregnancies were complicated by death of both infants. Four of the MC twins were monoamniotic; these pregnancies were excluded. Therefore, 96 MC twin pairs with at least one survivor were eligible for this prospective follow-up study. Perinatal mortality data of these twins will be reported separately in a large cohort, collected over a longer time period . A group of 98 dichorionic (DC) twins was matched for gestational age at delivery, gender, birth weight, ethnicity of the mother and study center.The study was approved by the institutional review board of the University Medical Center Utrecht, and local permission was obtained in the other centers. Study participation by written consent was obtained from the parent(s). All twins with identical gender (i.e. boy-boy and girl-girl twins) were identified from the electronical databases of each hospital. We checked the medical charts to assess chorionicity. Chorionicity was determined on the basis of first-trimester ultrasound assessment and/or postpartum pathological examination of placentas and intertwin membranes. The diagnosis of TTTS was made antenatally by standard ultrasound criteria Information on the occurrence of cerebral palsy (CP) was obtained from all children. The diagnosis of cerebral palsy (CP) was made according to standard criteria The participants were tested with the Griffiths Mental Developmental Scales Statistical analysis was performed with the SPSS 12.0 statistical package. Continuous variables were compared by independent Student t-testing if normally distributed or otherwise by means of Mann Whitney U testing. Categorical variables were compared by Fisher's exact test. Linear regression was used to estimate mean differences of the DQ-scores according to chorionicity and donor or recipient of a TTTS-twin. These mean differences were adjusted for possible confounders. Statistical significance was based on two-sided tests with a cut-off level of 0.05.During the study period 182 MC and 189 DC long-term survivors were approached for this prospective follow-up study. In forty-six twin pairs the developmental test could not be performed, either due to being lost-to-follow-up (15 MC and 20 DC infants) or parents did not approve participation  and one out of 189 long-term DC survivors had CP . Two MC infants had CP due to complications specific to MC twin pregnancies. In one case fetal death of the smaller co-twin occurred at 26 weeks of gestation most likely due to placental insufficiency, with subsequent very preterm delivery of the surviving twin. This boy had a severe intraventricular hemorrhage (grade III) with posthemorrhagic ventricular dilatation and atrophy of brain tissue and developed spastic diplegia. In the other case, the pregnancy was complicated by TTTS (Quintero stage III) with laser coagulation of the connecting placental vascular anastomoses. Both infants were delivered at 37 weeks of gestation. The recipient showed normal development in contrast to the donor twin. The antenatal ultrasound of the brain showed echogenicity. The MRI after birth showed generalized cerebral atrophy and signs of an old hemorrhage in the periventricular area and in the basal ganglia. This child had a quadriplegia with severe mental retardation. The other two MC infants with CP were born very preterm  and both developed spastic quadriplegia as a result of cystic periventricular leukomalacia (PVL). The cause of CP in the infant of the DC group (born at 31 weeks of gestation) is currently unknown, but may be genetically determined as this child also suffers from albinism. This child has diplegia with psychomotor retardation.After losses to follow-up, 140 MC infants and 142 DC infants without CP participated in this prospective follow-up study and were tested with the Griffiths Mental Developmental Scales. Baseline characteristics of the study population are shown in The majority of the tested infants showed normal development (98.2%). Five out of 282 long-term survivors had a mild developmental delay and two infants had a severe developmental delay. There was a significant difference in development between boys and girls. Boys had lower DQ scores  and the proportion of mildly delayed development was higher among male infants. Infants born before 32 weeks of gestation and/or with a birth weight <1500 gram had significantly lower DQ-scores than infants born after 32 weeks and/or ≥1500 gram . There was no significant difference between infants who were small-for-gestational age and infants with a birth weight above the tenth percentile.p = 0.423), which was mainly due to the higher proportion of mildly delayed hearing and speech development in MC twins. These results were in agreement with the parental impression of the development of their infant(s), as assessed by the HSCS-PS questionnaire (ionnaire .p = 0.109).Developmental outcome of the twins after an uneventful course of pregnancy  is shown in p = 0.338). This trend was found in both MC and DC discordant twins.Eighteen MC pregnancies were complicated by antenatal signs of TTTS (17.6%), of which six had been treated by laser coagulation of vascular anastomoses. Five pregnancies had been treated by serial amnioreduction. The other pregnancies did not receive any treatment, either due to low stage TTTS or immediate delivery after diagnosis. Three TTTS pregnancies were complicated by death of one of the twins. There were 33 long-term survivors, of which one had CP (3.0%). Twenty-seven long-term survivors were tested. All of them had a normal development . However+0 and 25+5 weeks, respectively), one IUD at 26+1 weeks was most likely due to placental insufficiency, and the other IUD remained unexplained (at 32+2 weeks' gestation). One DC fetal death at 31+1 weeks was caused by placental insufficiency and the other two deaths remained unexplained . Only one (MC) survivor developed CP, all other survivors showed normal development.In this cohort of 282 infants, four MC and three DC twin pregnancies were complicated by single intra-uterine death. In two MC cases IUD occurred after laser ablation for TTTS ; two CP-cases were due to complications specific to MC twin pregnancies  and two cases of CP as a result of cystic PVL after preterm birth. One DC twin had CP (0.5%) without clear cause. The majority of 2-year-old twins had a normal developmental status. There were no significant differences between MC and DC twins, apart from a slight delay in hearing and language development in MC twins. Birth weight discordancy did not influence long-term outcome, although there was a trend towards lower DQ-scores in the smaller twin than in its larger co-twin.The response rate of this follow-up study was 76%. This raises concern about the possibility of a non-response bias. In order to reduce this possibility to a minimum, we obtained information on neurological status and development from the attending pediatrician of the non-participants. In this way, we were able to report the study population based CP incidence. From five twin pairs no follow-up data were retrieved. We made an assumption that these children did not develop CP, since they did not have any major  complications during their neonatal period.A limitation of this study is that it was not population-based. The data in this study should thus be interpreted with care since a selection bias may have been introduced due to the specific nature of tertiary referral centres. However, long-term outcome of the twins was even better than reported in previous publications.With regard to the analyses based on the Griffiths test, it may be that our results are somewhat biased in favor of the DC group. That is, non-responders in the DC group were found to be born earlier than the responders in this group, a difference that was not found in the MC group, which may have led to an underestimation of developmental delay especially in the tested DC group. However, since we expected a worse outcome of MC twins due to possible cerebral damage in the presence of haemodynamic imbalance, this bias does not influence our conclusion that long-term outcome of MC twins was not shown to be worse than that of DC twins.However, it should be noted that neurological handicaps and mental retardation may become evident only several years after birth, so a new evaluation of CP and developmental delay at the age of five years is preferable.It is well known that the incidence of abnormal long-term neurodevelopmental outcome in MC pregnancies complicated by TTTS is high. After treatment with serial amniodrainage, most studies on long-term outcome report a 22–26% incidence of neurological impairment It is also well known, that the risk of neurological abnormality in the surviving twin after co-twin intra-uterine death is higher in MC than in DC twins. The mechanism leading to damage of the surviving sibling is an acute blood loss through the placental vascular anastomoses into the dying fetus, leading to hypovolemia in the survivor, which in turn might cause death from hypovolemic shock or cerebral ischemia due to hypoperfusion Intrauterine growth restriction (IUGR) is another important risk factor for cerebral palsy and impaired neurological development. Gratacos and co-workers p<0.05). TTTS was considered to be responsible for most cases of adverse outcome in the MC group. In the same period, Adegbite and collegues The increased risk of neurological disability in MC survivors of TTTS or co-twin fetal death suggests that cerebral injury in MC twins is related to the vascular anatomy of the placenta. Placental vascular anastomoses, which are nearly always present, can cause haemodynamic imbalance and subsequent ischemic injury to the brain. Cranial ultrasound abnormalities in the neonatal period also suggest that monochorionity itself is a risk factor for cerebral damage In summary, the majority of 2-year-old twins had a normal neurodevelopmental status. There were no significant differences between MC and DC twins. CP may occur more frequently in MC twins and was mainly due to complications specific to MC twin pregnancies (TTTS and co-twin death). Outcome of MC twins was favourable in the absence of TTTS or co-twin death. More studies are needed to evaluate the effect of improved obstetric management on the long-term neurodevelopmental status of ‘uncomplicated’ MC twins. Meanwhile we suggest a routine cranial ultrasound examination after birth and neurodevelopmental follow-up.


Acute pulmonary embolism (PE) is a common disease which frequently results in life-threatening right ventricular (RV) failure. High-risk PE, presenting with hypotension, shock, RV dysfunction or right heart thrombus is associated with a high mortality, particularly during the first few hours. Accordingly, it is important to commence effective therapy as soon as possible. In the case described in this report, a 49-year-old woman with myotonic dystrophy type 1 presented with acute respiratory failure and hypotension. Transthoracic echocardiography showed signs of right heart failure and a mobile right heart mass highly suspicious of a thrombus. Based on echocardiographic findings, acute thrombolysis was performed resulting in hemodynamic stabilization of the patient and complete resolution of the right heart thrombus. This case underscores the important role of transthoracic echocardiography for the diagnosis, management and monitoring of PE and underlines the efficacy and safety of thrombolysis in the treatment of PE associated with right heart thrombus. Right sided heart thrombi can be found in 4-18% of patients presenting with acute pulmonary embolism . They maHere, we present a case where transthoracic echocardiography was used for the diagnosis, management and follow-up of pulmonary embolism with right heart thrombus.A 49-year-old woman with myotonic dystrophy type 1  was admitted to our neurological intensive care unit because of respiratory failure due to myoneuronal hypoventilation and clinical suspicion of pneumonia. Due to progressive respiratory decompensation she had to be intubated prior to admission. At arrival, her vital signs were as follows: body temperature 37°C, blood pressure 130/70 mmHg, pulse 70 beats per minute (bpm) and respiratory rate 14 breaths per minute. Laboratory tests showed moderately elevated CRP  and normal white blood cell counts. Blood and urine cultures drawn at this time were ultimately found to be negative. D-dimer was elevated . Computed tomography (CT) of the chest was performed showing focal consolidations and ground glass opacity in the left lower lobe as signs of an evolving pneumonia. There was a filling defect in the right upper lobe pulmonary artery highly suggestive of a pulmonary embolus  and high doses of vasopressors.Bedside transthoracic echocardiography was performed immediately, showing a mobile mass 3 cm × 2 cm), highly suspicious of a thrombus, moving back and forth from the right atrium to the ventricle through the tricuspid orifice during cardiac cycle. The right ventricle was severely dilated combined with right ventricular mid free-wall hypo-/akinesis and nearly normal contraction of the apical segment (McConnell sign) . Tricusp cm, highin situ lysis than due to migration to the pulmonary arteries. Duplex ultrasonography of extremity veins revealed deep vein thrombosis of the left superficial femoral vein, profunda femoris vein and popliteal vein.Echocardiography on day two after thrombolytic therapy showed a complete resolution of the right sided heart thrombus Figure  See AddiHeparin-induced thrombocytopenia type II and activated protein C-resistance could be excluded.torsades de pointes-tachycardia [Myotonic Dystrophy Type 1 is an autosomal dominant multisystem disorder caused by a mutated expansion of a CTG repeat in the 3' untranslated region of a serine-threonine kinase gene on chromosome 19 . MD1 pathycardia .Acute pulmonary embolism (PE) is a common disease that may lead to life-threatening right ventricular failure. Computed tomography of the chest is the main diagnostic approach for the detection of PE. However, in a highly unstable patient as in our case - where transportation of the patient is not feasible - diagnosis of PE may be accepted solitary on the basis of direct or indirect echocardiographic findings. Additionally, duplex ultrasonography of the extremities - especially in the presence of central venous catheters - might be helpful to confirm the diagnosis.High-risk PE, defined by hypotension, shock or RV dysfunction, is associated with a high mortality, particularly during the first few hours. Accordingly, it is important to commence effective therapy as soon as possible. In stable patients, therapy with unfractionated or low-molecular-weight heparin is recommended. According to current ESC guidelines, thrombolytic therapy is the first-line treatment (unless there are contraindications for thrombolysis) in addition to heparin for patients with high-risk PE presenting with cardiogenic shock and/or persistent arterial hypotension .While therapeutic strategies for the treatment of high-risk PE are well defined, the optimal therapy for a right heart thrombus in PE - thrombolysis, heparin alone or embolectomy - remains an open and controversial debate. A meta-analysis published in 2002 points to an advantage of thrombolytic therapy in reducing mortality compared to anticoagulation with heparin or surgical embolectomy . A more Despite rapid recovery of right ventricular function after thrombolytic therapy only a slight improvement of systolic PAP was seen in our patient during the two weeks follow-up. However, this short term follow-up might not be representative for the long-term outcome regarding pulmonary arterial pressure. Ribeiro et al. reported in their echocardiography study of 78 patients with acute PE and increased PAP that a stable phase of systolic PAP is achieved within 38 days after diagnosis of acute PE. The treatment (heparin versus thrombolysis) received in the acute phase resulted in no significant difference in the time to achieve the stable phase or in the level of systolic PAP estimated for the stable phase. A systolic PAP of > 50 mmHg at the time of diagnosis of acute PE was associated with persistent pulmonary hypertension after one year .Our patient was diagnosed with massive PE according to clinical symptoms and echocardiography showing right heart failure and a right heart thrombus. Based on these findings, acute thrombolysis was performed resulting in hemodynamic stabilization of the patient and complete resolution of the right heart thrombus. Echocardiography was the only possible diagnostic approach as transportation of the patient for a CT scan was not feasible due to hemodynamic instability. Transthoracic echocardiography is a fast, practical and sensitive approach for the detection of right ventricular dysfunction and right heart thrombus. Therefore it should be performed as soon as possible in unstable patients with suspected PE.The present case underscores the important role of transthoracic echocardiography for the diagnosis, management and monitoring of PE. It also underlines the efficacy and safety of thrombolysis in the treatment of PE associated with right heart thrombus.Written informed consent was obtained from the patient's legal representative for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.CVP: central venous pressure; MD1: myotonic dystrophy type 1, Curschmann-Steinert Syndrom; PAP: pulmonary artery pressure; PE: pulmonary embolism; rt-PA: recombinant tissue plasminogen activator; RV: right ventricular; TAPSE: tricuspid annular plane systolic excursion; TTE: transthoracic echocardiogram.The authors declare that they have no competing interests.BH conceived the case report, performed bedside echocardiographic examinations, reviewed literature and wrote the manuscript. HD performed bedside echocardiographic examinations. LG, HD and ACB have been involved in drafting the manuscript. EMF provided radiologic assistance. AP contributed critical revision of the manuscript. GB has supervised and commented the manuscript. ACB was the supervisor of echocardiographic examinations, head of the echocardiography lab, and contributed by revising the manuscript critically. All authors read and approved the final manuscript.Computed tomography (CT) of the chest on admission. Contrast-enhanced CT scan of the chest showing a filling defect in the right upper lobe pulmonary artery (marked by arrow) indicative of a pulmonary embolus and bilateral pleural effusions; (A) transversal view, (B) coronal view.Click here for fileTransthoracic echocardiogram (TTE) on admission. Apical 4-chamber view.Click here for fileTTE with a right sided heart thrombus. Apical 4-chamber view with a mobile thrombus in the right atrium moving through the tricuspid orifice into the right ventricle.Click here for fileTTE on day 2 after thrombolytic therapy. Apical 4-chamber view shows complete disappearance of the right heart thrombus.Click here for file


Hepatitis C virus infection and interferon treatment may be associated with retinopathy but visual function is generally unaffected. This paper reports the rare occurrence of unilateral macular edema with visual loss. We present an interventional case report with fundus photograph and optical coherence tomography (OCT). A 48-year-old white male with hepatitis C, treated with a six-month course of pegylated interferon alpha and ribavirin, complained of gradual reduction in the vision of his left eye. Visual acuities were 20/16 right and 20/400 left with clinical examination and OCT confirming cystoid macular edema.This report shows that cystoid macular edema may rarely occur in association with hepatitis C infection and/or interferon therapy. Physicians and ophthalmologists should be alert to this potential but infrequent association as the resultant visual loss is a significant potential complication that should be discussed when obtaining informed consent for interferon treatment. Hepatitis C, virus (HCV) may be associated with an ischemic retinopathy thought to be secondary to HCV-induced vasculitis. ContempoHere we present optical coherence tomography (OCT) evidence of cystoid macular edema (CME) probably due to pegylated interferon alpha treatment for hepatitis C in an otherwise healthy individual with none of the common risk factors for CME. To our knowledge, only two similar cases of this rare and emerging phenomenon have previously been reported worldwide.8A 48-year-old white male was referred to the ophthalmic accident and emergency by his general practitioner. He gave a six-month history of gradual, painless loss of central vision in his left eye. Visual acuities were 20/16 in the right eye unaided and 20/400 best corrected in the left eye. Both eyes were white with no intraocular inflammation and left eye CME was confirmed on slit-lamp biomicroscopy. Furthermore, OCT showed three symmetric cystic spaces in the external plexiform layer, increased free space between the neurosensory epithelium and retinal pigmented epithelium and disorganization of internal retinal architecture at the macula -consisteHe was known to be a chronic carrier of HCV (diagnosed in 2004 and attributed to an episode of tattooing) and had undergone a standardized six-month treatment regimen of pegylated interferon-alpha-2a (Pegasys 18 mcg) subcutaneous injections and oral ribavirin 600 mg twice daily under the gastroenterology service of a neighboring hospital. The visual loss had started some weeks into his course, with no subjective improvement on completing the full six-month course, although his viremia was successfully cleared. He had no previous ophthalmic history such as trauma or operations and the left eye was not amblyopic. He was on no other medication and there was no other medical history, including diabetes or hypertension. He was a non-smoker.Topical treatment was commenced in the form of ketorolac 0.5% eye drops four times daily and dexamethasone 0.1% eye drops four times daily to the left eye. At the three-month follow-up he reported no subjective improvement in vision and acuity remained poor at 20/200 despite a repeat OCT scan showing resolution of edema. Fundus fluorescein angiogram and fundus autofluorescence confirmed macular retinal pigment epithelial atrophy. Active management and further follow-up were declined by the patient.The incidence of retinopathy associated with interferon treatment varies in literature from 18-86%.[There are only two similar reports published.8 The firHypertension and diabetes mellitus have been shown to be risk factors in the development or progression of interferon-associated retinopathy in HCV-infected individuals. However, neither was identified in our patient.In summary, we present a rare case of CME with attendant visual loss which may be causally related to interferon alpha treatment of hepatitis C. Ophthalmologists and physicians should be aware of the possible ocular side-effects of interferon and whilst treatment of the systemic disease must take priority, we suggest that the potential for transient or longer-term visual loss be discussed when obtaining informed consent for interferon therapy.


FTO (fat mass and obesity associated) gene have been associated with an increased risk of obesity. However, the function of its protein product has not been experimentally studied and previously reported sequence similarity analyses suggested the absence of homologs in existing protein databases. Here, we present the first detailed computational analysis of the sequence and predicted structure of the protein encoded by FTO.Genetic variants in the We performed a sequence similarity search using the human FTO protein as query and then generated a profile using the multiple sequence alignment of the homologous sequences. Profile-to-sequence and profile-to-profile based comparisons identified remote homologs of the non-heme dioxygenase family.Our analysis suggests that human FTO is a member of the non-heme dioxygenase (Fe(II)- and 2-oxoglutarate-dependent dioxygenases) superfamily. Amino acid conservation patterns support this hypothesis and indicate that both 2-oxoglutarate and iron should be important for FTO function. This computational prediction of the function of FTO should suggest further steps for its experimental characterization and help to formulate hypothesis about the mechanisms by which it relates to obesity in humans. FTO gene with an increased risk of obesity, characterized by an increase in body max index due to fat mass rather than lean mass that is seen in children as early as age seven [Two recent reports ,2 characFTO is unknown [FTO is crucial to guide the search for a mechanism relating this gene to obesity.However, the mechanisms by which this genetic variability relates to obesity remain obscure. These publications indicate that the function of  unknown  and that unknown . KnowledFTO is a member of the non-heme dioxygenase (Fe(II)- and 2-oxoglutarate-dependent dioxygenases) superfamily.Here we report evidence obtained by computational analysis indicating that the protein coded by Ostreococcus) and diatoms   we identified sequences homologous to human FTO in different eukaryote groups including vertebrates , green algae  members of the non-heme dioxygenase family were identified. Additionally, the secondary structure predictions of the FTO family showed high similarity with the known structures of AlkB, a member of the non-heme dioxygenase family [E. coli AlkB proteins (PDB entries 2iuw and 2fdi) matched the FTO N-terminal region with an E-Value of 3.2 × 10-21 and 3.1 × 10-12, respectively (estimated error rate < 3%) despite their low level of sequence identity to the human FTO protein (approximately 17%). The next match corresponded to the hypothetical protein TM0957 from Thermotoga maritima, however it was considered unreliable given its short length (28 amino acids) and high E-value (0.02).To investigate if fold recognition analysis would generate supporting results, we submitted the FTO N-terminal region as a query to an independent fold assignment system based on profile-profile comparisons (see Methods). The profiles generated for the human and FTO human gene) are members of the non-heme dioxygenase superfamily.Given the E-values of the HMMer searches, the reliability of secondary structure predictions, and the fold assignment results, we are confident that the proteins of the FTO family , a known member of the non-heme dioxygenase family, also has a wide phylogenetic distribution  and is ubiquitously expressed in all human tissues. HIF acts as a sensor of oxygen level and affects the expression of over one hundred genes . This moTo investigate if FTO could be acting in a similar manner, we studied its sequence using an algorithm for prediction of protein cellular localization . The reThe FTO human gene product has a predicted molecular mass of 50 KDa. With that mass it would need a Nuclear Localization Signal (NLS)  in orderIn light of these computational results we hypothesize that FTO is a sensor of the cell's metabolic state and when dysfunctional can result in an obese phenotype. We identify the N-terminal of human FTO as having a high likelihood of determining its cellular localization, which could be verified by mutational analysis.FTO relates to obesity and to accelerate the discovery of novel molecular therapies for this condition.Here we have provided valuable information about FTO by indicating its possible catalytic function, and we have pointed to the amino acids involved in cofactor (Fe) and co-substrate (2-oxoglutarate) binding in human FTO as well as in its homologous proteins in other organisms, which could be used as models for the study of the human disease. This insight should help to guide experiments to clarify the mechanisms by which We first performed BLAST sequence similarity searches  using thProfiles of the alignment as global hidden Markov models (HMMs) were generated using HMMer . ProfileFold recognition analyses were performed using profile-to-profile comparisons of the HMM profile of the FTO family to profiles generated for each sequence of known structure with its homologues . The2OG (2-oxoglutarate)AlkB (Alkylated DNA repair protein)ESTs (Expressed sequence tags)FGENESH (Find Genes using HMM)FTO (fat mass and obesity associated)HIF (Hypoxia-inducible factor)HMMs (Hidden Markov Models)JGI (Joint Genome Institute)NBCI NLS SNPs (Single nucleotide polymorphisms)LSP carried out the initial sequence and structural analysis of the domain. LSP and MAA interpreted the data and prepared the manuscript. All authors read and approved the final manuscript.


T-cell epitopes that promiscuously bind to multiple alleles of a human leukocyte antigen (HLA) supertype are prime targets for development of vaccines and immunotherapies because they are relevant to a large proportion of the human population. The presence of clusters of promiscuous T-cell epitopes, immunological hotspots, has been observed in several antigens. These clusters may be exploited to facilitate the development of epitope-based vaccines by selecting a small number of hotspots that can elicit all of the required T-cell activation functions. Given the large size of pathogen proteomes, including of variant strains, computational tools are necessary for automated screening and selection of immunological hotspots.Hotspot Hunter is a web-based computational system for large-scale screening and selection of candidate immunological hotspots in pathogen proteomes through analysis of antigenic diversity. It allows screening and selection of hotspots specific to four common HLA supertypes, namely HLA class I A2, A3, B7 and class II DR. The system uses Artificial Neural Network and Support Vector Machine methods as predictive engines. Soft computing principles were employed to integrate the prediction results produced by both methods for robust prediction performance. Experimental validation of the predictions showed that Hotspot Hunter can successfully identify majority of the real hotspots. Users can predict hotspots from a single protein sequence, or from a set of aligned protein sequences representing pathogen proteome. The latter feature provides a global view of the localizations of the hotspots in the proteome set, enabling analysis of antigenic diversity and shift of hotspots across protein variants. The system also allows the integration of prediction results of the four supertypes for identification of hotspots common across multiple supertypes. The target selection feature of the system shortlists candidate peptide hotspots for the formulation of an epitope-based vaccine that could be effective against multiple variants of the pathogen and applicable to a large proportion of the human population.. It is a new generation computational tool aiding in epitope-based vaccine design.Hotspot Hunter is publicly accessible at  The binding of fragments of processed antigens by major histocompatibility complex (MHC) molecules of antigen presenting cells (APCs) and their presentation to T-cells is crucial for immune surveillance and defence against bacteria, parasites, viruses and tumors. Recognition of the MHC-restricted target peptide on the surface of APCs by the surveying T-cells of the immune system is mediated through the T-cell receptors (TCRs) ,2. PeptiChlamydia trachomatis outer membrane protein [It can be postulated that an epitope-based vaccine ideally should contain a minimal number of epitopes that cover a vast majority of the human population -10. T-ce protein . These c protein .Nearly all existing publicly available promiscuous T-cell epitope prediction servers perform prediction for a single protein sequence per submission, while those that accept multiple sequences are not tailor-made to predict hotspots; they rely on manual visualization techniques. Given the large size of pathogen proteomes, it is painstaking to integrate the individual prediction results, and therefore impractical to use these tools for large-scale systematic study of promiscuous epitopes, which is necessary for a global view of the localizations of the epitopes in the proteome and analysis of their antigenic diversity. Herein, we present Hotspot Hunter, a web-based computational system for large-scale screening and selection of candidate immunological hotspots in pathogen proteomes through analysis of antigenic diversity . It alloIt has been reported that combining predictions by several methods results in greater accuracy -19. A so. The HLA-DR training dataset had 2396 (448 binders and 1948 non-binders) 9-mer peptides related to six HLA-DRB1 variants . The training data was from experiments conducted on 340 15-mers to measure their binding affinity to each of the six HLA-DR alleles. The 15-mers came from three protein sources: MEL40 , SSP2 , HCV . The transformation from 15-mers to 9-mers was performed utilizing SYFPEITHI motifs [The predominant length of peptides that bind HLA class I molecules  is nine amino acids . HLA claThe ANN and SVM models employed herein were the same as those used in MULTIPRED1 . Three-lA SVM with Gaussian kernel (g = 0.1 and c = 0.5) was used for HLA-A2 and Gaussian kernel (g = 0.1 and c = 2) for HLA-A3 supertype . SVMs wiet al. [Candida infection in human, showed that Hotspot Hunter accurately identified two of the three HLA-DR restricted immunological regions experimentally identified using PBMC proliferation and IL-2 ELISpot assays [The prediction performances of models for HLA-A2 and -A3 supertypes were validated using experimental results from a systematic study of human papillomavirus type 16 E6 (NCBI Accession: P03126) and E7 (NCBI Accession: P03129) proteins . Two of et al.  conducteet al.  showed tet al.  and the et al. ,35. Hotset al.  and hepaet al. . All thet assays ..Each input protein sequence to Hotspot Hunter is truncated into overlapping 9-mer peptide sequences with an 8 amino acid overlap and analysis is carried out on each individual 9-mer. To predict immunological hotspots (regions of high concentration of 9-mer promiscuous binders), we have developed two scoring schemes for HLA class I and class II supertypes. The scheme for HLA class I supertypes is based on high scoring individual 9-mers within a window of 30 amino acids ,33, and The web interface of Hotspot Hunter is intuitive, making use of a set of simple Graphical User Interface forms. Programs were built using a combination of Perl, CGI and C language. The implementation was carried out in SunOS 5.9 UNIX environment.Analysis of antigenic regions in a single protein is initiated by opening the "Single Sequence Query" page of Hotspot Hunter, the query protein sequence is pasted into the query box, a name is assigned and the HLA supertype of interest is selected. The input protein sequence should be in FASTA format and at least 15 (for HLA-DR supertype) or 30 (for HLA-A2/A3/B7 supertype) amino acids long and at most 2000 amino acids long. Input sequences that contain symbols other than amino acids  or are of lengths outside the acceptable length range, are not be processed – an error message will be displayed. The sequence pasted in the query box is treated as one single protein sequence (carriage returns are ignored). Figure . Predictions are performed on each sequence in the alignment and gaps in the sequences are removed prior to the prediction. However, the gaps are still displayed on the results page and, therefore, hotspots can appear on the results page interspersed by gaps of variable sizes, depending on the alignment. The results page provides a global view of the localizations of the hotspots in the proteome set, enabling analysis of antigenic diversity and shift of hotspots across protein variants. An example of the Hotspot Hunter analysis of a set of dengue virus type 1 capsid sequences is shown in Figure "Multiple Sequences Query" function allows users to predict hotspots in a set of protein sequences by submitting a multiple sequence alignment file either in ALN  or PHY (PHYLIP) format  summarizes the process of decision-making in selecting the best consensus hotspot and best candidate targets. The output of this analysis is a comprehensive report, which facilitates interpretation of results and the selection of validation experiments. A sample of the target selection analysis output for dengue virus type 1 NS2b protein is provided in Figure "Target Selection" function for multiple sequence queries employs a computational method to identify candidate peptides for the formulation of an epitope-based vaccine that could be effective against multiple variants of the pathogen and is also applicable to a large proportion of the human population. The predicted peptides from consensus hotspots of aligned protein sequences are analyzed for selection of best targets for further experimental validation. All the predicted consensus hotspots are ranked to aid in the decision support, aiming to select the best consensus hotspot. Figure S4 on Hotspot Hunter FAQ page Hotspot Hunter performs predictions on four HLA supertypes, HLA-A2, -A3, -B7 and -DR. It allows integration of the prediction results of the four supertypes for identification of hotspots common across multiple supertypes, which is facilitated by the function "Hotspots Common Across Supertypes". The user needs to upload the output files (generated by "Multiple Sequences Query") of the supertypes of interest for the same set of sequences. Hotspots common across any combination of the four supertypes can be analysed.Experimental approaches for identification of T-cell epitopes are laborious and costly, and thus are not applicable for large-scale screening across multiple HLA alleles and pathogen proteomes. Several publicly available online computational systems have been developed for the prediction of peptides binding to HLA alleles and supertypes, such as SYFPEITHI  based on and CandiVF – Candida albicans Virulence Factor Database [Hotspot Hunter is different from other online servers in several aspects. First, the prediction engine combines the strengths of the ANN and SVM methods for better and more robust prediction performance; second, it presents users with predicted immunological hotspots, which are regions of high concentration of predicted promiscuous HLA binding peptides; third, it is suitable for systematic studies of a large set of pathogen proteomes as it can concurrently analyze multiple sequences and present a map providing a global view of their localizations in the proteome, which is the main novelty of the system; and finally, the system provides a utility for selecting candidate experimental targets based on antigenic diversity analysis. Therefore, Hotspot Hunter is a new generation computational tool aiding in epitope-based vaccine design. A customized version of Hotspot Hunter has been integrated into some of our in-house specialized databases, such as the Tumor Antigen Database Database , for preHLA – Human Leukocyte Antigen; MHC – Major Histocompatibility Complex; ANN – Artificial Neural Network; SVM – Support Vector Machine; APC – Antigen Presenting Cell; TCR – T-cell Receptor; NCBI – National Center for Biotechnology Information; MOG – Myelin Oligodendrocyte Glycoprotein; IEDB – Immune Epitope Database and Analysis Resource; TAP – Transporter Associated Protein; ER – endoplasmic reticulum; HCV – Hepatitis C Virus; PBMC – Peripheral Blood Mononuclear Cell.The authors declare that they have no competing interests.in silico experiments and drafted the manuscript. KNS, ATH, KXL, JTA and VB participated in the design of the study. VB conceived the study, participated in its design and coordination and helped draft the manuscript. JTA, VB and CKK critically reviewed the manuscript. All authors read and approved the final manuscript.GLZ and AMK performed the


Arabidopsis thaliana has increased tremendously during the last years. These resources permit the database-assisted identification of putative cis-regulatory DNA sequences, their binding proteins, and the determination of common cis-regulatory motifs in coregulated genes. DNA binding proteins may be predicted by the type of cis-regulatory motif. Further questions of combinatorial control based on the interaction of DNA binding proteins and the colocalization of cis-regulatory motifs can be addressed. The database-assisted spatial and temporal expression analysis of DNA binding proteins and their target genes may help to further refine experimental approaches. Signal transduction pathways upstream of regulated genes are not yet fully accessible in databases mainly because they need to be manually annotated. This review focuses on the use of the AthaMap and PathoPlant® databases for gene expression regulation analysis and discusses similar and complementary online databases and web-tools. Online databases are helpful for the development of working hypothesis and for designing subsequent experiments.The number of online databases and web-tools for gene expression analysis in  Its small genome is completely sequenced and contains only a low amount of repetitive DNA and a high gene density [http://arabidopsis.org [ density . Furtherpsis.org . The larAll scientific questions that address developmental processes or biotic and abiotic signal response reactions focus on the understanding of gene expression regulation. There are three levels of regulation, pre-transcriptional, transcriptional and post-transcriptional. The pre-transcriptional level concerns chromatin-structure and remodelling. Transcriptional control is executed mainly by transcription factors (TFs) that recruit the transcriptional preinitiation complex to the promoter. A major aspect of post-transcriptional regulation is RNA stability affected by small RNAs. Arabidopsis thaliana more than 1500 TFs were initially identified which constitute at least 5% of all protein coding genes [Out of these levels, transcriptional control is the most accessible level for database-assisted analysis . Transcrng genes ,5. More ng genes . Experimental data on these TFs varies significantly. While usually a few type members of each family have been extensively analysed, the function of all family members often remains unknown. The same applies for information on the binding site of these factors. For members of 25 TF families representative binding sites have been published and were annotated to databases -9. cis-regulatory sequence in the promoters of genes which will then be a target site of a TF that regulates expression of the gene through DNA binding [A simple concept for transcriptional control is based on the presence of the binding site or  binding . A next  binding . cis-regulatory elements, predictions can be established about which TF family is involved in regulating transcription [Based on the occurrence or combination of cription . Howevercription .cis-regulatory elements recognized by known interacting TFs colocalize in their target genes [Often, single TFs are not sufficient for the regulation of gene expression . To gainet genes . Furtheret genes . Arabidopsis thaliana and the genomic identification of their target sequences may reveal which genes are subjected to small RNA-mediated degradation [Another level of complexity that can be addressed with databases is the post-transcriptional control of gene expression. A large number of small RNAs have been cloned from radation . Arabidopsis thaliana. It will focus on two databases, AthaMap and PathoPlant and the questions that can be addressed with them. The subjects discussed are schematically shown in the flow chart in Fig. (1). To address these subjects, also other online resources are available which are summarized in Table (1). This review describes internet resources that are available for the study of gene expression regulation in 2Arabidopsis thaliana. AthaMap was initially developed by matrix-based sequence searches using alignment matrices derived from several binding sites of the same TF [6 predicted binding sites from 103 plant transcription factors [AthaMap is a database that generates a genome-wide map of putative transcription factor binding sites (TFBS) for  same TF . Subsequ same TF . Current factors . Online  factors . Combina factors . This fu factors .3Arabidopsis thaliana subjected to pathogen infections, signal components, and elicitors [cis-regulatory elements.PathoPlant is a database on plant-pathogen interactions and signal transduction reactions . The datlicitors . Web-too4cis-regulatory sequence and/or transcription factor(s) that regulate(s) gene expression through this region .The experimental analysis of gene expression may still include a classic deletion and reporter gene approach to identify a regulatory region in a gene. This may delineate a region that is essential for gene expression. A next step aims to predict the specific For this purpose the region of interest can be displayed in AthaMap and putative TFBS that occur in this region can be predicted . This alA. thaliana specific databases such as AGRIS, ATHENA, and ATTED-II (Table 1) can be employed to predict cis-regulatory sequences [® databases (Table 1) to display either TFBS or cis-regulatory sequences that have been experimentally described before but for which no binding TF has been predicted [If the regulatory region is found in a promoter region, also other equences -20. Furtredicted -23. 51B). Such combinatorial elements will also be displayed in AthaMap if they were previously annotated [cis-regulatory elements or TFBS occur in the experimentally defined regulatory region it can be experimentally investigated if more than one is required for gene expression. In many cases a single TFBS does not cause a specific expression unless in combination with other regulatory elements. A database-assisted approach could answer the question if a regulatory sequence is part of a known combinatorial element . For this a web-tool to identify combinatorial elements in the AthaMap database can be employed [1). Furthermore, another tool to identify genomic positions of previously determined cis-regulatory sequences is PatMatch available at TAIR [1).If TFBS predicted by database-assisted analysis were experimentally confirmed to be relevant for gene expression, it may be interesting to find out if other genes harbour this combination of TFBS in a similar arrangement . A list of genes identified with AthaMap that harbour TFBS under investigation can be exported to the PathoPlant database to investigate if the genes are coregulated [Another problem that can be addressed with web-resources is the identification of conditions under which genes with similar TFBS are coregulated . These are for example TAIR [Several web-based services harbor gene expression data from ple TAIR ,26, AtGeple TAIR ,28, NASCple TAIR , Stanforple TAIR ,31, Botaple TAIR , Gene Exple TAIR , and Genple TAIR . Arabidople TAIR ,35, BAR ple TAIR , the Comple TAIR , and Genple TAIR  allow co8cis-regulatory elements starts with the identification of coregulated genes . For example PathoPlant allows the identification of coregulated genes and the export of the gene list to AthaMap for subsequent TFBS identification [1) will identify combinatorial patterns of TFBS in a set of user-provided Arabidopsis genes. The analysis platform uses a matrix-based approach to identify TFBS in promoter regions and analyses the results with respect to specific patterns of TFBS that are overrepresented compared to a set of control genes [A different approach to identify fication . The comol genes .1). For example the Regulatory Analysis Tools (RSA tools) offer the possibility to submit a set of promoter sequences to identify common motifs [A. thaliana genes. The tools available online may also be downloaded and implemented locally. For example the Binding-site Estimation Suite of Tools (BEST) includes four commonly used motif-finding programs: AlignACE, BioProspector, CONSENSUS and MEME and the optimization program BioOptimizer [Considering the fact that not all TFBS of predicted TFs are known, it may also be of interest to find patterns of conserved sequence motifs in a set of coregulated genes. For this purpose several online tools are available (Table n motifs . A similn motifs . Promomeptimizer .1) may be used to query databases of known motifs with new motifs derived from similar sequences [Since these programs often yield sequence motifs derived from similar sequences that are conserved in a set of sequences, it may be of interest to find out if the motifs have been identified previously as regulatory sequences or TFBS. For this, STAMP  [Arabidopsis thaliana small RNA transcriptome screening [A large number of internet resources are available for gene expression analysis in  in vivo . Further in vivo . To learvivo [1) . AthaMapcreening . mRNA tr1). With the application of massive parallel signature sequencing (MPSS) approaches, the analysis of gene expression is recently undergoing a revolutionary development . With th1) comprises 10 distinct subcellular locations, >6743 non-redundant proteins and represents the proteins encoded in the transcripts responsible for 51% of Arabidopsis expressed sequence tags [Arabidopsis thaliana yet, but a recent review on plant proteome analysis summarizes all Arabidopsis proteome articles published in 2006 [Ideally not only transcriptome data will be integrated into databases but also proteome data . Proteinnce tags . Another in 2006 .in vivo TFBS, transcription factor and target gene expression and on cellular localization of transcripts and proteins.An integrated gene expression database of the future will contain data on functional


The aim of this study is to determine the prevalence of anti-HCV among injecting drug users (IDUs) in Lebanon, to establish the current prevalence of HCV genotypes in this population and to determine whether demographic characteristics and behavioral variables differ between participants who were HCV-RNA positive and those who were HCV-RNA negative or between the different genotypes. Participants were recruited using respondent-driven sampling method. The blood samples were collected as dried blood spots and then eluted to be tested for HCV, HBV and HIV by ELISA. Anti-HCV positive samples were subjected to RNA extraction followed by qualitative detection and genotyping.Among 106 IDUs, 56 (52.8%) were anti-HCV-positive. The two groups did not differ in terms of age, marital status, and nationality. As for the behavioral variable, there was a trend of increased risky behaviors among the HCV-RNA positive group as compared to the HCV-RNA negative group but none of the variables reached statistical significance. Half (50%) of the 56 anti-HCV-positive were HCV-RNA positive. Genotype 3 was the predominant one (57.1%) followed by genotype 1 (21%) and genotype 4 (18%).The predominance of genotype 3 seems to be the predominant genotype among IDUs in Lebanon, a situation similar to that among IDUs in Western Europe. This study provides a base-line against possible future radical epidemiological variant that might occur in IDUs. The prevalence of HCV antibody (anti-HCV) among intravenous drug users (IVDUs) who share injecting equipment exceeds 50% ,2. This Hepatitis C virus has been classified into 6 major genotypes (1-6) and into several subtypes  ,8. The gThe aim of this study is to determine the prevalence of anti-HCV among injecting drug users (IDUs) in Lebanon, to establish the current prevalence of HCV genotypes in this population and to determine whether demographic characteristics and behavioral variables differ between participants who were HCV-RNA positive and those who were HCV-RNA negative or between the different genotypes.Participants included in this study were part of a bio-behavioral surveillance study done to assess HIV prevalence among four vulnerable groups . Two well established Lebanese non-governmental organizations (NGOs) that work with IDUs helped in the study.Participants in this study were recruited using a relatively new technique entitled respondent-driven sampling (RDS) that has been shown to be effective in reaching difficult to reach or invisible populations for which there is not sampling-frame . A totalPeripheral blood was collected by a finger prick with a single use Lancet and then blotted onto a high quality filter paper (Schleicher & Schuell 903). The blood spot was allowed to air dry to be used as a dried blood spot (DBS) for further testing at the Molecular Biology Research Laboratory at the Faculty of Health Sciences, American University of Beirut (AUB).For each serological test, one DBS disc was cut and placed in the well of a flat-bottomed uncoated micro plate. The blood was then eluted as described previously by Parker and Gublit .Eluates were tested for anti-HCV by ELISA using a modified protocol for Ortho HCV 3.0 SAVe . Anti-HCV positive DBS samples were then subjected to RNA extraction using QIAamp MiniElute Virus Spin (QIAGEN) for further qualitative HCV detection and genotyping.COBAS AMPLICOR Hepatitis C Virus Test, version 2.0 (v2.0) was used for qualitative in vitro diagnostic HCV detection in the RNA extracted samples using COBAS AMPLICOR Analyzer (Roche). Qualitatively positive HCV aliquots of denatured amplicon by Cobas Amplicor were genotyped using a single LINEAR ARRAY HCV Genotyping strip (LINEAR ARRAY Hepatitis C Virus Genotyping Test - Roche) which is coated with a series of oligonucleotide probes specific for various HCV genotypes.Descriptive statistics including means and standard deviation and frequency distributions were computed for demographic variables and behavioral variables. Comparisons between HCV-RNA positive and HCV-RNA negative groups were performed using the Chi-squared test for categorical variables  and t-test for continuous variables (for example age). Significance was set at the 5% level. All analysis were done using SPSS version 16.0 .Only 28 (50%) of the 56 anti-HCV-positive were HCV-RNA positive. Characteristics and behaviors of those who were HCV-RNA positive and HCV-RNA negative are summarized in Table Only HCV genotypes 1, 3 and 4 were detected and one sample showed mixed genotype (1 and 3). The distribution of genotypes is shown in Table Characteristics of the HCV RNA-positive sample population (n = 28) according to their genotype is shown in Table HCV-RNA positivity (50%) detected in our anti-HCV-positive IDUs patients is in agreement with similar studies from different parts of the world including Europe,16, NortThe distribution of HCV genotypes in our IDUs showed that HCV genotype 3 was the predominant genotype (57.1%) followed by genotype 1 21.4%) and genotype 4 (17.9%). This was surprising and in contrast with our earlier findings where genotype 4 was the predominant genotype (47.5%) in Lebanese patients . The str1.4% and Since IDUs become infected with HCV in the early phases of drug addiction and most become exposed repeatedly to the virus through needle-sharing, it is expected that infection with more than one genotype would be common . Mixed iData on major HCV genotypes circulating among injecting drug users in neighboring Middle Eastern countries is still limited ,22. ThisDespite the small number of patients investigated, it can be concluded that genotype 3 seems to be the predominant genotype among IDUs in Lebanon, a situation similar to that among IDUs in Western Europe. However, the authors would like to acknowledge that the sample method used (RDS) was not random as is the situation for population-based surveys such as the National Health and the Examination Survey and thus our findings may or may not be representative of the entire population of IDUs in Lebanon. This study provides a base-line against possible future radical epidemiological variant that might occur in IDUs.The authors declare that they have no competing interests.2 designed the study. KK3 carried out the laboratory tests and have been involved in drafting the manuscript and revising its content. SS performed the laboratory tests. SR was responsible for drafting and finalizing the manuscript and has given final approval of the version to be published. All authors read and approved the final version.ZM have made contributions to acquisition, analysis and interpretation of data. KK


N-linked glycosylation is a major mechanism for minimizing virus neutralizing antibody response and is present on the Human Immunodeficiency Virus (HIV) envelope glycoprotein. Although it is known that glycosylation changes can dramatically influence virus recognition by the host antibody, the actual contribution of compartmental differences in N-linked glycosylation patterns remains unclear.env gp120 C2-V5 region and analyzed 305 clones derived from plasma and other compartments from 15 HIV-1 patients. Bioinformatics and Bayesian network analyses were used to examine N-linked glycosylation differences between compartments. We found evidence for cellspecific single amino acid changes particular to monocytes, and significant variation was found in the total number of N-linked glycosylation sites between patients. Further, significant differences in the number of glycosylation sites were observed between plasma and cellular compartments. Bayesian network analyses showed an interdependency between N-linked glycosylation sites found in our study, which may have immense functional relevance.We amplified the in vivo. These findings could also have relevance in shielding and evasion of HIV-1 from neutralizing antibodies.Our analyses have identified single cell/compartment-specific amino acid changes and differences in N-linked glycosylation patterns between plasma and diverse blood leukocytes. Bayesian network analyses showed associations inferring alternative glycosylation pathways. We believe that these studies will provide crucial insights into the host immune response and its ability in controlling HIV replication  Variable regions (V1-V5) in env are spaced between the conserved regions (C1-C5). Both N-linked and O-linked glycans are present on the HIV envelope glycoprotein. O-linked glycans are present on several unidentified serine or threonine residues in env gp120, but very little is known about their actual role in governing the viral phenotype of both HIV and simian immunodeficiency virus (SIV) [env gp160 [env gp120 region [The HIV-1 envelope  from 15 HIV-1 infected patients on HAART, each displaying different levels of plasma viremia and T cell counts. In addition to the patient specific changes observed in our analyses, we further found evidence in favor of compartmental NLG differences and distinct cell-specific molecular changes in the env C2-V3 region of HIV-1.We analyzed 305 clones of HIV-1 variants derived from plasma and diverse blood leukocytes , CD4Phylogenetic tree reconstruction using a maximum likelihood heuristic search algorithm showed patient-specific clustering of virus from all compartments, confirming the patient origin of HIV-1 variants and the absence of cross-patient contamination. Further, within each patient there was distinct clustering of HIV-1 sequences from each compartment, confirming the purity of diverse blood leukocytes and plasma samples  at position 279, whereas the PBMC and plasma-derived sequences were found to have aspartic acid (D) at the same position. The amino acid lysine (K) was uniquely seen in monocyte-derived HIV sequences at positions 335 and 350, whereas other compartments showed arginine (R) at that position. Further amino acid differences across compartments were found at positions 320, 336, 360 and 440 as illustrated in Table + T cell and plasma-derived sequences at position 360. However a lost of significance was found (p = 0.22) when we correct for multiple comparisons.From our signature pattern analysis, we found seven single amino acid differences from the 305 HIV-1 sequences across five different compartments  in our inter-patient analysis. Among the NLG sites identified, the one at position 338 from patient 13 was unique to the HIV-1 strain and had not previously been recorded in the Los Alamos HIV database.N-linked glycosylation analysis using the N-Glycosite program  identifi2 test for the comparison of frequencies across five different compartment using a 5 × 2 contingency table gave a p-value of 0.0015 for position 295. Fisher's exact test for the comparison of frequencies observed from plasma versus all cell-types  showed significant difference (p = 0.04) in the frequencies observed at position 448. We are aware that these values might be affected by our treatment of several clones from the same patient and from the same compartment, as the different sequences are not truly independent events. Hence, we extended this analysis further to include only the median number of NLG sites per patient per compartment and not each individual count of NLG sites per sequence. While this procedure eliminates the effect of non-independence, it also lessens the number of observations and possibly the statistical power. When we analysed the median number of NLG sites between compartments with the gap-stripped sequences  of functional relevance described by Wei et al [et al [et al [To deduce the possible biological and functional relevance of these findings, we also compared the 17 potential NLG sites found in our study to functionally analyze N-linked glycosylation sites published by Wei et al . Most ofei et al   we have account for. This pitfall was overcome by combining the Bayesian network analysis with a bootstrap approach. The strength of the arcs is proportional to its bootstrap support and not to the the importance of the conditional independencies to the joint probability of the network. Overall, these analyses have allowed us to obtain a detailed profile of potential N-linked glycosylation distribution and possible inter-relationship between NLG sites in HIV-1 in vivo. Such adaptation includes the modulation of N-linked glycosylation and cell-specific single amino acid changes in HIV. Given the importance of the envelope glycoprotein in neutralization, pathogenesis, tropism and viral evasion, we analysed the C2-V5 region of 305 HIV-1 env gp120 sequences from plasma and diverse blood leukocytes of 15 patients on HAART. Single amino acid differences specific to plasma and cellular compartments were observed. Notable was the presence of lysine (K) in monocytes at positions 335 and 350 whereas these positions have arginine (R) in the other compartments. This suggests that positions 335 and 350 have the greatest need to maintain a basic amino acid residue (lysine) in monocytes, compared with the other compartments. This difference may have a role in viral adaptation to monocytes and possibly relevant to monocyte tropism. In addition, the isoleucine at position 360 was unique to CD4+ T cells (p < 0.044). Additionally the presence of valine was unique to PBMC (and absent in plasma and the three other cell types) at position 360. Although the monocyte compartment is a subset of the PBMC compartment, the majority of PBMC sequences were found with valine (V) and the monocyte sequences with alanine (A). We believe that although all the cellular compartments we analyzed were derived from the whole PBMC, the valine may be specific to a leukocyte subset, which was not analyzed in this study, due to the limitation of human bleed obtained from each patient. Nonetheless, this difference between monocytes and PBMC supports our observation that compartmental-specific amino acid changes in HIV-1 env gp120 are present. Our results agree with previous work on single amino acid changes in the env gene in association with viral tropism and pathogenicity [et al [in vivo environments.In order for HIV to be successful in evading the immune system, both cell-free and cell-associated forms of HIV must adopt distinct molecular strategies to adapt and survive genicity . A recen+ T cells and plasma in our HAART patient pool, glycosylation change in the virus may enable it to overcome the selection pressures from the antiretroviral regimen. Further examination of the NLG sites using Bayesian networks found unique associations between sites 301–444, 293–362, 295–334, and 301–332. These associations could indicate alternative pathways for glycosylation and possible simultaneous co-selection of glycosylation sites. Similar to the study of evolutionary interactions between NLG sites by Poon Art F, Y [ex vivo-collected cells from each patient, our results reflect the closest possible snapshot to the in vivo situation.The modulation of N-linked glycosylation sites in the HIV-1 envelope has been known to facilitate viral evasion from the host immune system. Similar to the signature pattern analysis, the N-linked glycosylation analysis yields interesting results showing compartmental variations and possible associations among glycosylation sites. An examination of the NLG frequencies along the C2-V5 region  between plasma and cellular compartment sequences. This is consistent with the notion that the number of glycosylation sites is highly conserved in HIV-1 [env. Despite the loss of statistical significance when we applied the Bonferroni correction for multiple comparison from P = 0.022 to P = 0.115, we believe that it is still an important finding together with the rest of our statical analysis. This is because by correcting for multiple comparisons, we are also increasing the risk of making a type II error which might lead us to not report a correlation when there is one. Between both gap-stripped and gap-inclusive analysis, the gap-inclusive alignment gives us the actual number of glycosylation sites, as no glycosylation sites were omitted in the evaluation. Not finding any significant differences in this gap-inclusive alignment indicates that possible selective pressure to remove glycosylation sites from the more conserved part of env was compensated by the creation of new glycosylation sites in those parts of env that better tolerate substitutions, insertions and deletions.We examined the variation in the number of glycosylation sites among compartments (gap-free and gap-inclusive) to infer a global picture of HIV-1 in HIV-1 . The disin vivo, it is important to note that the NLG observed in our sequences are distinctly patient-specific (p ≤ 2.2 × 10-16). This supports our belief that the selection of NLG sites are primarily dependent on the patient's immune response. The choice to examine 305 sequences from 15 independent patients with a range of CD4+ T cell counts and plasma viral load in our study has allowed us to infer a balanced macroscopic perspective of HIV-1 adaptation in vivo during therapy. These analyses are the first to provide a detailed comparison of cell-free and cellassociated virus, especially in individual leukocyte types. Previous studies by Hanna et al [et al [in vivo and these data may have relevance in HIV immune recognition, viral adaptation, vaccine strategies and HIV pathogenesis in general.Even though we found evidence for compartmentalization of HIV-1 across plasma and diverse blood leukocyte populations na et al  and Lin in vivo. Further, this enhance our understanding of pathogenous differences between cell-free and cell-associated HIV-1. Consequently, these analyses will allow further biological and functional assessment of such molecular changes in the context of viral escape, adaptation and reservoir establishment in vivo. A better understanding of diverse N-linked glycosylation sites and their functional role may provide useful strategies for choosing and eliminating the "right" N-linked glycosylation site(s), thus facilitating the design of more effective envelope-based immunogens that elicit broad neutralization antibody responses.This study examined single cell/compartment-specific amino acid variations and unique differences in N-linked glycosylation patterns between plasma and diverse blood leukocytes. It has provided deeper insights into how HIV may evade antibodies and maintain its pathogenic potential. Bayesian analysis has shown associations that suggest possible glycosylation pathways. We believe that these analyses provide useful insights into the host immune response and its ability in controlling HIV replication This work is carried out in accordance with the human ethics guidelines and scientific principles set out by the National Health and Medical Research Council of Australia (NHMRC). All patients have given written consent on the study and understand that the study will be conducted in a manner conforming to the ethical and scientific principle set out by the NHMRC.Fifteen HIV-1 infected patients receiving HAART from the Westmead Hospital in Sydney, Australia, were enrolled in this study after prior consent. These patients exhibited varying plasma viral loads and T cell counts as shown in Table + T cells, CD8+ T cells and CD14+ monocytes) were separated from PBMC using magnetic beads coated with monoclonal antibodies  using the procedure described and developed by Potter et al [env gene. Reverse transcription-PCR (RT-PCR) of HIV-1 RNA extracted from plasma using the Qiagen RNA Extraction Kit  was carried out to amplify viral populations from the cell-free plasma fraction [+, CD8+ T cells, monocytes and plasma were cloned. In each case, the major amplicon population without cloning was first obtained to assess diversity within a patient. Following that, cloning and sequencing were performed as described previously [+ T cells was not successful for patient 6 and 9; neither was it successful for monocytes for patients 6, 7, 9, 14 and 15 or PBMC for patients 14 and 15. A total of 305 HIV-1 cloned sequences were generated from 15 patients. BLAST searches and phylogenetic analyses  [All 305 HIV-1 nucleotide sequences from the C2-V5 region of the (EMBOSS) . From th(EMBOSS) ) and verPhylogenetic reconstructions were performed to confirm the purity of viral sequences at the level of individual patients and each compartment analyzed. Phylogenetic analysis was performed based on the gap-stripped amino acid alignment using maximum likelihood on the ProML program (version 3.66) of the PHYLIP package  with the+ T cells, CD8+ T cells, monocytes and plasma) and compared inter-compartmentally. Given the extensive inter-strain/inter-patient variation in our 305 sequences, the majority consensus parameter was used. The "no fixed rates" option was chosen because a fixed rate would not capture the range of diversity observed.Signature pattern analysis was performed on the translated protein sequences using the Viral Epidemiology Signature Analysis (VESPA) software . The VESenv gp120 protein to examine for NLG differences between plasma and cell types  virus in vivo. Our data was analysed using the program N-Glycosite [N-linked glycosylation analysis was performed on all 305 protein sequences from the C2-V5 region of the lycosite , availablycosite   was examined. The χ2 test was used to compare the frequencies observed across five different compartments for each NLG site identified. A 5 × 2 contingency table was used to evaluate their statistical significance. Next, Fisher's exact test was used to compare the frequencies observed from plasma versus all cell-types together for each NLG site. We further analysed the differences in the mean and median number of NLG sites both across compartments and between patients using the non-parametric Kruskal-Wallis test, as the hypothesis of a normal distribution for the number of glycosylation sites was rejected by the Shapiro-Wilks test. All statistical analyses were performed with the R package [env gp120 protein sequences from different blood leukocyte populations and plasma in vivo.The frequency of NLG sites in the sequences found in both plasma and diverse leukocytes  consensus sequences from our clones. These five consensus sequences for each patient were then used for the Bayesian network analysis. This approach allowed us to remove any false positive associations caused because multiple cloned sequences from the same patient of the same compartment are likely to be highly similar. If the consensus sequences were not used, the arcs of the network could be artificially strengthened and we might overestimate of the significance of some associations. The most probable Bayesian network is the one that maximizes the posterior probability of the model given the data, subject to a prior distribution of model, which we assumed was uniform. We used a simulated annealing heuristic to search in the space of all possible Bayesian network structures [For each patient, we created compartmental  declare that they have no competing interests.YSH: Carried out the entire study, designed the framework, developed in-house bioinformatic tools for analysis and wrote the manuscript; ABA, MC, KD, KT and AMV provided highly coordinated help with statistical analysis and Bayesian network analysis; DD provided patients and their enrolment for this study, and provided all the clinical information needed and NKS conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.