Drosophila melanogaster female sperm storage is anecessary step in the reproductive process. Amo is a homolog of the humantransient receptor potential channel TRPP2 , which ismutated in autosomal dominant polycystic kidney disease. In flies Amo isrequired for sperm storage. Drosophila males with Amomutations produce motile sperm that are transferred to the uterus but theydo not reach the female storage organs. Therefore Amo appears to be amediator of directed sperm motility in the female reproductive tract but theunderlying mechanism is unknown.Sperm have but one purpose, to fertilize an egg. In various species includingin vivo imaging of dual labelled sperm to demonstratethat Drosophila sperm navigate backwards in the femalereproductive tract. In addition, we show that sperm exhibit hyperactivationupon transfer to the uterus. Amo mutant sperm remaincapable of reverse motility but fail to display hyperactivation and directedmovement, suggesting that these functions are required for sperm storage inflies.Amo exhibits a unique expression pattern during spermatogenesis. Inspermatocytes, Amo is restricted to the endoplasmic reticulum (ER) whereasin mature sperm, Amo clusters at the distal tip of the sperm tail. Here weshow that flagellar localization of Amo is required for sperm storage. Thisraised the question of how Amo at the rear end of sperm regulates forwardmovement into the storage organs. In order to address this question, we usedAmo is part of a signalling complex at the leading edge of the sperm tailthat modulates flagellar beating and that guides a backwards path into thestorage organs. Our data support an evolutionarily conserved role for TRPP2channels in cilia. Drosophila melanogaster, female sperm storage is critical formaximal reproductive success Drosophila female during mating but ∼80% areexpelled from the uterus when the first egg is laid The evolutionary success of sexual reproduction depends on the ability of motilesperm cells to find an egg and fertilize it. Different animals have evolved distinctand elaborate mechanisms to accomplish this essential task but female sperm storageis a commonly used strategy in species ranging from insects to mammals. InDrosophila males that arelacking the seminal fluid protein Acp36DE are inefficiently transferred from theuterus to the sperm storage organs There is relatively little known about the factors that govern sperm storage in thefemale reproductive tract. In flies, male accessory gland proteins contribute tothis process and sperm derived from Drosophila melanogasterPKD2) PKD2 result in autosomal dominant polycystic kidney diseasein vivo is poorlyunderstood Drosophila males with amo mutations aresterile; they produce motile sperm that are transferred to the uterus but the spermdo not reach the female storage organs We have previously shown that Amo is a sperm enriched protein that is essential forfertility and sperm storage in Drosophila sperm mightswim tail first. We used in vivo imaging of dual labelled sperm todemonstrate that wild type Drosophila sperm do in fact navigatebackwards in the female reproductive tract. In addition, we discovered that spermexhibit activated flagellar beating upon transfer to the uterus. Amo mutant spermremain capable of reverse motility but fail to display hyperactivation, suggestingthat activated flagellar beating is a requirement for sperm storage in flies.In the present study we investigate the role of Amo in sperm motility within thefemale reproductive tract. We show that although Amo is expressed in the endoplasmicreticulum (ER) during earlier stages of spermatogenesis, it is the flagellarlocalization at the tip of the sperm tail, which is critical for making spermstorage competent. This finding coupled with Amo's role in directional spermmovement prompted us to hypothesize that Drosophila males with Amo mutations produce motile sperm thatare transferred to the uterus but do not reach the female storage organs   , and the protein .In contstorage . Althougstorage , it was storage , suggest rescued . Since bDrosophila sperm swimbackwards in the female reproductive tract in vivo. Thispattern of directed motility has not been reported for sperm of any otherspecies.The requirement for Amo at the flagellar tip coupled with its pivotal role indirected sperm motility prompted us to ask how Amo could regulate head firstmovement? We hypothesized that sperm might travel in reverse or tail firstrather than in a forward direction. In order to explore this possibility, weadapted methods that allowed us to assay directional sperm movement within thefemale reproductive tract in real time Drosophila sperm and Amo might act as asensor at the leading edge, serving a distinct pathfinding function. In order totest these possibilities, we generated amo mutant fliesproducing dual labelled sperm and mated these males to wild type female. Wefound that amo mutant sperm still are capable of backwardsswimming, both in the uterus and in the seminal receptacle channels in ciliary structures. In Drosophila sperm travel backwards in the uterusand seminal receptacle. As far as we know, this type of motility has not beenreported for sperm of any other species. One potential rationale for this type ofdirectional movement is that it would permit sperm to “back in” to thefemale storage organs. This is supported by analyses of sperm organization withinthe female seminal receptacle Amo's unique distribution in the flagellum led us to investigate the behaviourof sperm in the female reproductive tract. Our studies reveal several novelfindings. First, we used real-time analysis of sperm expressing red and greenfluorescent proteins in the head and tail respectively to show that shortly aftermating, wild type Drosophila sperm acquirehyperactivated flagellar beating upon transfer to the female uterus. This is similarto mammalian sperm capacitation, a process that “switches on”spermatozoa, thus rendering them capable of fertilizing an egg Secondly we show that wild type Amo is a logical mediator of sperm motility in Drosophila. In addition to itsfavourable localization in the sperm tail, TRPP2 proteins are calcium permeablenon-selective cation channels In summary we show that Amo localization defines a unique niche at the leading edgeof sperm, which are traveling tail first. Activation of Amo serves to modulateflagellar beating and guides a backward trajectory into the sperm storage organs. Inkeeping with an evolutionarily conserved sensory role for TRPP2 channels in cilia wepostulate that Amo is ideally located to receive cues upon transfer to the femalereproductive tract. The nature of the stimuli to which the TRPP2 channel complexresponds remains a matter of investigation. But in the light of our data and recentevidence from vertebrate models it is tempting to speculate that ligands rather thanmechanical cues are critical for triggering TRPP2-mediated signalling Amo knockout flies (1amo) have beendescribed amo, (CG6504) and approximately 1 kb of 5′ flankingsequence amo (CG6504) genomic rescue construct wasgenerated by site-directed mutatgenesis using standard procedures. We sequencedall constructs to verify that no errors were introduced by PCR. Flies expressingthe wild type and mutant genomic rescue constructs were crossed into theamo null background for analysis of fertility, spermstorage and sperm dynamics. Flies expressing Protamine-B-labelled with redfluorescent protein (ProtB-DsRed) on the third chromosome were kindly providedby John Belote and a line expressing don-juan-GFP (dj-GFP) on the thirdchromosome was obtained from Barbara Wakimoto ® 488 antibodies were used for visualization. Sperm tails were stained with Alexafluor®-594 conjugated concavalin A  and sperm heads by 4′,6-Diamidin-2-phenylindol(DAPI). Images were recorded using a Zeiss LSM510 confocal microscope .Dissection and preparation of testis and sperm as well as the anti-Amo antiserum(1∶3000) have been described 1118w (wt) virgin females were performed for5 days. At that time both parents were removed from the vial. The number ofprogeny that eclosed from each vial was counted. Ten vials were scored for eachgenotype.Males of various genotypes were separated upon eclosion and maintained inisolation 3 days prior to mating. Single pair matings with1118w females and mating wasinterrupted after 20 minutes. To analyze the beat frequency, sperm were releasedfrom a seminal vesicle or from a mated-uterus into a Petri dish containingHEPES-buffered saline solution -1-piperazineethanesulfonic acid (HEPES), pH 7.4). Seven areasaround the sperm mass were recorded. Three sperm tails per area were analyzed.One hundred fifty frames were acquired per area at a frame rate of 50 Hz. Spermbeating was recorded on an inverted microscope  witha charge-coupled camera device .Beat frequency was analysed using ImageJ software .Three-day old virgin male and/or female flies were used for these studies. Maleswere mated to −/−amo background) and virgin1118w females were separated for four daysprior to mating. Immediately after termination of mating, female flies wereanaesthetised with CO2 and dissected in HEPES-buffered salinesolution using fine forceps. The lower reproductive tract was removed withoutcompression of the uterus by gently severing the ovipositor from surroundingcuticle and longitudinally opening the mid-ventrum. The digestive tract wassevered close to the anus and the ovaries were removed at the base of thelateral oviducts. Female reproductive tracts were aspirated using a Pasteurpipette and transferred to a 15 µm slide 2×9 well , containing 70 µl HEPES-buffered salinesolution per well. Real-time imaging of dual-colour fluorescently labelled spermin the female reproductive tract was performed on a microscope ZEISS LSM 510 DUOequipped with a LD LCI Plan-Apochromat 25x/0.8 glycerine objective . Microscopic analysis was started withinfive minutes after unsolicited termination of mating. Excitation of thefluorophores (GFP and DsRed) was performed at 489 and 532 nm, respectively. Forsimultaneous detection of the red and green fluorescence, the LSM 5 Liveconfocal scanner was used, collecting emitted fluorescence in the range495–525 nm for the dj-GFP fusion protein, and 560–675 nm forProtB-DsRed. 5000 images were recorded per sample at 15 frames per second with512×512 pixels and a pixel dwell time of 112 µs. . Image analysis andsperm tracking were performed using Imaris tracking software .Freshly hatched males . Unpairedstudent's t-Test was used for statistical analysis between two groups.Analysis of sperm dynamics and subcellular localization of TRPP2 wild type andmutant transgenes was performed in a blinded fashion.Figure S1Sperm Storage Organs Dissected From Females Mated With Wild Type or−/−amoFlies. A. Schematic model of the female reproductive system.Ovary (ov), spermathecae (sp), seminal receptacle (sr), uterus (ut).B–F. Seminal receptacles dissected30–60 minutes after observed mating. Wild type virginfemales were mated to males of different genotypes as indicated:B. Wild type, C.amo−/−,D.amo−/−;P[amo],E.amo−/−D627V],;P[amoand F.amo−/−D627V];P[amo/amo.(TIF)Click here for additional data file.Figure S2Amo Localizes to the Endoplasmic Reticulum (ER) inSpermatocytes. A. Intracellular localization of Amo inspermatocytes . B. Expressionpattern of the ER marker PDI-GFP in spermatocytes. C. Mergedimages.(TIF)Click here for additional data file.Figure S3Amo Expression by Western Blot Analysis. Lysates were preparedfrom male flies of various genotypes and subjected to immunoprecipitationwith anti-Amo antisera. Western blots were probed with anti-Amoantisera.(TIF)Click here for additional data file.Figure S4D627VTransheterozygous Sperm.Subcellular Localization of Amo in Amo/Amo Immunofluorescent labeling of sperm ofthe genotypeamo−/−D627V];P[amo/amo. A. DAPI.B. Concavalin A. C. Anti-Amo. D.Merged image. Scale bar 20 µm.(TIF)Click here for additional data file.Figure S5Analysis of Sperm Speed in the Uterus. Frequency distribution ofsperm speed in the female reproductive tract .(TIF)Click here for additional data file.Figure S6D627V.Absence of a Dominant Negative Effect of AmoFertility tests using heterozygous+/amo−mutant males show that introduction of one (dark grey bars) or two copies(light grey bars) of transgenic AmoD627V (3rdchromosome) does not result in impaired fertility.(TIF)Click here for additional data file.Video S1Wild Type Sperm in the Female reproductive Tract. Live imagingof wild type sperm bearing dj-GFP and ProtB-DsRed transgenes in the femaleuterus and seminal receptacle. Green fluorescent protein  and red fluorescent protein  were detectedsimultaneously. Isolated sperm head movement (white) in the uterus and theseminal receptacle is also demonstrated.(MOV)Click here for additional data file.Video S2−/−Amo in theFemale Reproductive Tract. Live imaging of−/−amo sperm bearing dj-GFPand ProtB-DsRed transgenes in the female reproductive tract. Sinceamo−/− sperm donot reach the storage organs, there are no sperm visualized in the seminalreceptacle. There is only background autofluorescence detected. IsolatedSperm head movement (white) in the uterus is also demonstrated.(MOV)Click here for additional data file.Video S3Single Sperm Movement in the Female Reproductive Tract. Liveimaging of a single wild type sperm labelled with dj-GFP and ProtB-DsRedtransgenes in the seminal receptacle is demonstrated. The sperm moves tailfirst.(MOV)Click here for additional data file.Video S4Single Sperm Movement ofamo−/−Sperm in the Female Reproductive Tract. Live imaging of asingle amo−/− spermlabelled with dj-GFP and ProtB-DsRed transgenes. The sperm movesbackwards.(MOV)Click here for additional data file.Video S5Tracking of Sperm in the Female Reproductive Tract.Representative tracking of sperm heads in the uterus. Each sperm head(magenta) is marked with a dot. The pseudocolor bar at the right representsthe time of tracking during the experiment.(MOV)Click here for additional data file.Video S6Wild Type Sperm in the Female Reproductive Tract. Real-timeimaging of dual-colour fluorescently labelled wild type sperm in the seminalreceptacle and uterus.(MOV)Click here for additional data file.


This report details the case of 17 year old identical twins who both presented with paroxysmal supraventricular tachycardia (PSVT). Electrophysiological studies revealed atrioventricular nodal reentry tachycardia (AVNRT) in both twins. Successful but technically challenging slow pathway ablation was performed in both twins. This is the first reported case of confirmed AVNRT in identical twins which adds strong evidence to heritability of the dual AV node physiology and AVNRT. A review of the current literature regarding PSVT in monozygotic twins is provided. Paroxysmal supraventricular tachycardia (PSVT) caused by accessory pathways or dual atrioventricular node (AVN) physiology were initially thought to be randomly occurring congenital anomalies.,2 HoweveIdentical female twins were referred with palpitations from a young age. Twin A was first seen in clinic at the age of 13 years. She had been experiencing rapid palpitations since the age of 12. Her palpitations were triggered by activity and occurred roughly x 4-5/month. The episodes were associated with breathlessness and presyncope. Most episodes were terminated with vagal maneuvers with only one episode required intravenous adenosine. Physical exam and echocardiography of twin A were unremarkable. Her resting 12 lead ECG was normal bar left axis deviation. During tachycardia, the12-lead ECG during palpitations showed a narrow complex tachycardia (rate = 180 bpm) with retrograde negative P waves in lead II about 20 ms off the onset of the QRS. Oral metoprolol (25 mg twice daily) reduced the frequency of the episodes but did not abolish them and hence the decision for management with catheter ablation.Twin B was first seen in clinic at the age of 11 years of age. She had experienced palpitations once every 2-3 months since the age of 10. Again the episodes were related to exertion and were associated with shortness of breath and presyncope. The episodes were terminated by lying flat. Examination, echocardiography and resting 12-lead ECG were normal bar left axis deviation. On this occasion, SVT was diagnosed by an external event recorder. Initial management was with vagal maneuvers only until her palpitations became more frequent at the age of 17 when more invasive management was sought.A standard 4 wire study was performed in both patients: decapolar CSL response to coronary sinus from the left subclavian vein, johnson curve quadrapolar catheters to high right atrial and right ventricular apical positions, and a 4 mm non-irrigated tip ablation catheter initially in the His position .Twin A: Basic intervals were within normal limits  applications. Many of the RF applications had to be curtailed due to fast junctional tachycardia with dissociated atrial and ventricular activity . After tTwin B: Baseline intervals were normal ; i.e. a re-entrant origin. PSVT occurs with an incidence of 35 per 100,000 people a year [The major causes of PSVTs are AVNRT (~60%), and AV re-entrant tachycardia (AVRT) (~ 30 %) with smaller contributions from intra-atrial re-entry, micro re-entrant AT and sinus nodal re-entry .PSVT caused by AV accessory pathways or dual AVN physiology were formerly thought to be randomly occurring congenital anomalies due to pathological substrates present from birth.,2 HoweveBack in 1976, Mispireta et al [A similar clinical scenario was reported by Bennett et al . Here idIn more recent studies investigating familial clustering of pre-excitation syndromes, an autosomal dominant pattern of inheritance has been suggested -9. Even The possibility of inheritable AVNRT has been suggested by a couple of case reports. Lu et al demonstrated bystander dual AVN physiology in 16-year-old identical female twins who both have AVRT caused by the same left lateral AV accessory pathway .In the case presented here, the presence of dual nodal physiology with symptomatic AVNRT, similar retrograde jumps in VA conduction  and simiThese 2 instances could justify the presence of AV dissociation during manipulation of the catheters in the slow pathway region or application of RF in the same area.The unique case presented here adds evidence to the inheritability of AVNRT as previously suggested. Further studies of AVNRT involving genetic mapping in correlation with the delineation of the AV nodal arterial supply and histological presence of inferior nodal extensions may be warranted.


It is well known that migraine attacks can be precipitated by various stimuli. More than 50% of migraine with aura (MA) patients know of at least one stimulus that always or often triggers their MA attacks. The objective of this study was to expose MA patients to their self-reported trigger factors in order to assess the causal relation between trigger factors and attacks.We recruited 27 MA patients who reported that bright or flickering light and/or strenuous exercise would trigger their migraine attacks. The patients were experimentally provoked by either different types of photo stimulation, strenuous exercise or a combination of these two factors. During and following provocation the patients would report any aura symptoms or other migraine related symptoms.Of 27 provoked MA patients 3 (11%) reported attacks of MA following provocation. An additional 3 patients reported MO attacks. Following exercise, 4 out of 12 patients reported migraine, while no patients developed attacks following photo stimulation.Experimental provocation using self-reported natural trigger factors causes MA only in a small subgroup of MA patients. Prospective confirmation is important for future studies of migraine trigger factors and in the clinical management of migraine patients.


Introduction: The standard non-muscle invasive bladder cancer (NMIBC) endoscopic diagnosis suffers from the frequently unsatisfactory white light evaluation accuracy leading to residual lesions being left behind. Monopolar transurethral resection of bladder tumors (TURBT) is marked by a substantial morbidity rate requiring further amelioration. Methods: Small size tumors (under 1 cm) are feasible for “en bloc" resection. Bipolar TURBT was defined as being able to challenge the gold-standard status of monopolar resection due to the reduced complication rates. Plasma-button vaporization was introduced as a promising large bladder tumors’ ablation modality. Narrow band imaging (NBI) cystoscopy became an increasingly popular method of enhancing tumor detection. Results: The “en bloc" resection of small size or thin pedicle tumors provides the conditions for avoiding tumoral tissue scattering. Bipolar resection is characterized by decreased perioperative bleeding risks and faster patient recovery. Plasma-button vaporization gained confirmation as an innovative approach, able to dispose large tumor bulks under complete control while minimizing the associated morbidity. NBI cystoscopy is a useful tool in identifying CIS lesions, small papillary tumors or extended margins of large tumor formations. As a cost-free technique, it may be extensively used both during the NMIBC initial diagnostic as well as during follow-up evaluation protocol. Conclusions: Having in mind the various modalities of ameliorating the bladder cancer diagnostic and treatment, NMIBC management should be tailored in accordance with the particularities of each case. Despite the technological advances achieved in the field of endourology, non-muscle invasive bladder cancer (NMIBC) continues to represent a challenging pathology, generally characterized by disappointing clinical outcomes and alarmingly high recurrence rates. The main reasons behind the relatively unsuccessful NMIBC therapeutic management are represented by the clearly doubtful oncological safety of the transurethral resection of bladder tumors (TURBT) [3] and, despite numerous attempts of improvement, continued to constitute the standard NMIBC treatment alternative during the past 50 years [4]. Moreover, the bladder mucosa endoscopic inspection under simple white light was often described as a rather blunt tool in detecting urothelial malignant lesions, thus creating the premises for leaving behind the future “recurrences" [5]. From this perspective, TURBT has been characterized as based on the “incise and scatter" oncologically flawed principle . Our experience during the past 5 years confirmed these findings and imposed bipolar TUR as a standard of “good clinical practice" in NMIBC treatment. Bipolar energy is most certainly recommended as the future of TURBT by the growing experience and increasingly numerous reports available in literature [8]. Subjectively speaking, we noticed that, during bipolar TURBT, a cleaner tissue cut is achieved, thus enabling a more accurate and precise tumor ablation to be performed. This feature is particularly useful during the resection of the thinned tumoral bed area, of tumors located on the lateral  or posterior bladder walls implying a high perforation risk as well as in cases of extensive flat lesions widely spread out throughout the bladder mucosa surface. The main advantages of bipolar resection over the monopolar approach are well defined by the published trials: reduced perioperative bleeding risks, superior haemostatic abilities and faster postoperative recovery  but eventually failed to gain acknowledgement in clinical trials. During the past 5 years, the plasma-button vaporization became increasingly popular as an efficient benign prostatic hyperplasia (BPH) treatment and was eventually oriented towards large bladder tumors’ management as well [10]. Endoscopic monopolar vaporization of tumors was at some point introduced as a viable NMIBC therapeutic alternative [11]. From the very beginning, we noticed that the bipolar plasma vaporization technique is able to provide the conditions for a fast, safe and efficient malignant tissue ablation phenomenon to be achieved. In our view, the BPV-BT cases selection criterion was represented by patients preoperatively diagnosed as presenting at least one apparently non-muscle invasive bladder tumor over 3 cm in diameter based on abdominal ultrasound, computer tomography and flexible WLC . The fact that it did not require the instillation of an expensive contrast agent (otherwise the main drawback of the photodynamic diagnostic – PDD) represented a substantial advantage from the very beginning. Narrow band imaging (NBI) cystoscopy was developed as a complementary method to the standard WLC, aimed to improve the bladder tumors’ detection solely based on an optic technological advancement [14]. Therefore, the vascular structures appear dark brown (capillary vessels) and green (veins), thus contrasting with the pink or white background of normal mucosa [15]. Subjectively, NBI technology was shown to accurately underline the specific vascular architecture of urothelial lesions, thus creating the impression of a three-dimensional visualization of tumors and clearly defining their limits [16].In short, the NBI visualization mode is based on the white light being filtered into 2 discrete centre wavelengths for blue (415 nm) and green (540 nm), otherwise strongly absorbed by hemoglobin and only able to penetrate the tissue surface [17] as well as to our own published data [18], NBI cystoscopy was proved to overcome the standard WLC diagnostic accuracy by finding more tumors. Patients with carcinoma in situ (CIS) lesions particularly benefited from the respective technique , which displayed a substantially increased detection rate in comparison with the white light evaluation [19].  Essentially, both according to literature . In such situations, NBI cystoscopy displayed superior detection rates over WLC in a randomized clinical setting, brought a significant contribution to an improved oncological outcome and reduced medium term recurrence rates being eventually obtained [12].  Moreover, according to our protocols, a special indication for the NBI evaluation is represented by cases of large bladder tumors (over 3 cm), widely acknowledged as associating hardly visible extended tumoral margins  2007-2013, financed from the European Social Fund and by the Romanian Government under the contract number POSDRU/107/1.5/S/82839".


Visual snow has a major impact on patients' quality of life. They describe persistent, dynamic, black & white tiny dots in the entire visual field (main criterion). Additional symptoms might be present. The literature on this condition is scarce -3 and thTo describe the clinical phenotype of patients with visual snow.Preliminary additional criteria for visual snow were generated retrospectively based on an internet-survey. By using telephone interviews, we re-tested these criteria prospectively and collected further information.The survey-data of 120 patients with self-reported visual snow was reviewed to generate preliminary criteria. These were re-tested by interviewing 115 patients. The main criterion  was met by 57 patients. Additional visual symptoms were excessive floaters (84%), persistent after-images (83%), "hard time seeing at night" (65%), "little cells that travel on a wiggly path" (77%), photophobia (70%), "moving objects leave trails" (56%), "swirls, clouds or waves with eyes closed" (51%), and bright flashes (51%). Requiring at least one or three of these additional criteria reduced the sensitivity by 2% and 7%. No patient described the visual symptoms consistent with persistent visual aura in migraine. A history of migraine was seen in 54%, with 35% having typical migraine with aura. None of the patients noted intake of illicit drugs prior the onset of visual snow. All ophthalmology tests were non-contributory.(i) Visual snow is a unique clinical syndrome. The main criterion  is almost always associated with at least three additional criteria. (ii) The visual symptoms are distinct from migraine with aura. (iii) The high prevalence of history of migraine (with aura) points to a susceptibility for visual snow in patients with migraine. (iv) Intake of illicit drugs and ophthalmological diseases may not be of pathophysiological relevance.


Sedimentaion rate is often used to manage pediatric patients with rheumatologic disease. Most management decisions are dependent on studies which have used Wintrobe or Westergren sedimentation rate methodologies. However, these methods suffer from the need for relatively large amounts of blood and long turn-around times. Determination of sedimentation rate using laser kinetic rate determination has allowed calibration to Westergren methods, low volume of blood needed for testing and very rapid results. We sought to compare the Wintrobe method to the ESR Stat method  to determine suitability of the ESR Stat method for patient testing.We performed a prospective comparison between the traditional Wintrobe and ESR Stat sedimentation rates in consecutive pediatric patients at a tertiary care pediatric hospital. Wintrobe and ESR Stat sedimentation data was fitted using a logarithmic model. Outliers were defined as those samples with ESR Stat sedimentation rates greater than 80 mm/hr and Wintrobe sedimentation rates less than 30 mm/hr . Retrospective chart review was performed on all patients undergoing testing.2 = 0.7768) and is seen below. Of interest was the identification of four patients who had apparently normal or mildly elevated sedimentation rates by the Wintrobe method versus an extremely high (greater than 120 mm/hr) by the ESR Stat method. These patients were noted to have lupus or lupus-like syndrome and a history of hemolytic anemia. Non-outlier samples were from patients who did not this combination of disease morbidities.A total of 131 pediatric patients (with one patient undergoing repeat testing because of sedimentation rate discrepancy) were tested. Age range was 18 months to 34 years with 29% being male. A logarithmic model appeared to best fit the data (RDiscrepancies in Wintrobe and ESR Stat sedimentation rates may identify a subgroup of with lupus (or lupus-like syndrome) and a history of hemolytic anemia. Careful consideration of methodology is needed when sedimentation rate testing is performed on pediatric lupus patients.Edward C. Wong: None; Deborah Yuan: None; Claas Hinze: None; Lawrence Jung: None.


The synergy between synaptic and postsynaptic firing dynamics in shaping neuronal encoding has not been explored. We show that by matching short-term synaptic dynamics with postsynaptic firing class, either stimulus rate or a rate change are encoded. The result may be relevant to understand the function of cortical microcircuits.Short-term synaptic dynamics and firing dynamics of neurons can each be classified into two types. In short-term plasticity, type 1 synapses show release-dependent depression and constant rate of recovery. They appear to encode a stimulus rate change. Type 2 synapses show release-independent depression and faster recovery at higher stimulus frequencies. They can follow the stimulus rate [We investigate if different combinations of synaptic and postsynaptic dynamics can shape neuronal encoding.Using NEURON, we simulated biophysically realistic cells with class 1 or 2 firing dynamics  that recSpiny stellate cells in the layer IV microcircuit preferentially receive inputs from type 1 synapses while star pyramidal neurons receive type 2 inputs . Even th


The authors report a case of metastatic gastrointestinal adenocarcinoma to the cervical spine in a patient with acquired immunodeficiency syndrome (AIDS) being treated with antiretroviral therapy. The source of this tumor could not be identified despite a thorough evaluation. A 49-year-old male being treated for AIDS presents with worsening neck pain and left distal arm weakness. MRI demonstrated an erosive mass within the cervical four vertebral body extending through the pedicle on the left side. Patient underwent needle biopsy followed by combined anterior and posterior fusion procedures. Pathology demonstrated metastatic gastrointestinal adenocarcinoma without known primary origin. He is currently undergoing palliative radiotherapy. This is an unusual case of metastatic gastrointestinal adenocarcinoma to the cervical spine. This should be included on the differential diagnosis of spinal lesions in this patient population and may represent a unique tumor in patients with HIV/AIDS who are on immunosuppressive therapy. The authors report an unusual case of symptomatic metastatic gastrointestinal adenocarcinoma without known primary tumor to the cervical spine in a patient with AIDS on chronic antiretroviral therapy.We report the case of a 49-year-old homosexual male being treated for AIDS who presented with worsening neck pain and left distal arm and hand weakness. MRI demonstrated an erosive mass within the C4 vertebral body extending through the pedicle on the left side and causing severe spinal stenosis Figures  and 2. APatient underwent needle biopsy followed by anterior cervical corpectomy and fusion and finally posterior lateral mass instrumentation and fusion . PatholoThe patient was successfully treated for post-operative cerebrospinal fluid collection in the neck with a lumbar drain. His neurological examination returned to its baseline. His CD4 counts remained stable preoperatively and postoperatively. He is currently undergoing palliative radiotherapy with 37.5 Gy over 15 fractions to his cervical spine.AIDS-defining cancers, such as Kaposi's sarcoma, non-Hodgkin's lymphoma, and cervical cancer, are quite common in patients with end-stage AIDS –3. Over  Some authors have argued that there is an association between chronic AIDS and human immunodeficiency syndrome (HIV) infection and the occurrence of colonic malignancy . StudiesAbout 10% of all cancer patients develop metastases to the spine , 5. AmonRavalli et al. noted in their seminal report three patients with HIV in less than one year who developed gastrointestinal carcinoma and suggested an increased frequency in this population . GastroiThis case stimulates interest in a possible association between AIDS/HIV, long-term antiretroviral therapy, and metastatic gastrointestinal adenocarcinoma without a clear primary site. We wonder if this particular metastatic gastrointestinal tumor is a unique tumor of the gastrointestinal system that is associated with chronic HIV/AIDS or chronic immunotherapy. In conclusion, this pathology must be kept on the differential diagnosis list in this patient population and further cases must be documented to clearly confirm this association.


Procalcitonin (PCT) has been studied extensively as a marker of sepsis; however, its clinical utility remains uncertain . Alternan = 87) were enrolled if they fulfilled the 1992 Consensus Statement [n = 31), with 71 healthy controls (HC) for comparison. Blood samples were collected within 24 hours of the surgical procedure or upon admission to ICU for sepsis patients. PCT was measured using a commercially available assay kit (Brahms PCT) and GE assessed using Affymetrix GeneChip Human Exon 1.0 ST arrays, where a set of biomarkers were identified a priori. A Support Vector Machine algorithm was used to calibrate the molecular biomarkers with respect to specific clinical groups. Bootstrap and permutation tests were performed on the difference in area under the receiver operating characteristic curves (AUC ROC) for PCT and crossvalidated posterior probabilities.The dataset was derived from two clinical trials conducted across four tertiary care settings in Australia between 2008 and 2011 (ACTRN12610000465055). Critical care patients , respectively. The differentiating ability of PCT and the SeptiCyte® Triage biomarker set is demonstrated in Figure The SeptiCyteIn comparison with the diagnostic performance of PCT, a limited set (<10) of molecular biomarkers potentially has a significantly better sensitivity and specificity profile for the detection of sepsis within a mixed systemic inflammatory patient group.


Marine salterns are artificial multipond systems designed for the commercial production of salt by evaporation of seawater. We report here the metagenomic sequence of the prokaryotic microbiota of a pond with intermediate salinity  of a saltern located in Isla Cristina, Huelva, southwest Spain. Haloquadratum walsbyi and the bacteroidete Salinibacter ruber  on July 2010. The sample was sequentially filtered through 5.0- and 0.22-µm-pore size polycarbonate filters (Millipore) as previously described . Total eSequences related to 16S ribosomal RNA genes were identified using the Ribosomal Database Project (RDP) . In addiEuryarchaeota (~84%) was the most abundant phylum present in this sample, followed by Bacteroidetes (~8%) and Gammaproteobacteria (~7%), suggesting a decrease of the microbial diversity in salt ponds of salterns when the salinity reaches salt concentrations >20%, being more similar to those found in crystallizer ponds than those found in intermediate-salinity ponds with <20% salts.Further metagenome analysis will contribute increased understanding of the genetic diversity present in marine salterns and particularly in ponds with intermediate salinities where the environmental conditions limit the presence of halophilic bacteria and enhance the growth of haloarchaea.SRP029970.The sequences obtained in this project have been deposited in the NCBI Sequence Read Archive under the accession no.


Genomic selection (GS) involves selection decisions based on genomic breeding values estimated as the sum of the effects of genome-wide markers capturing most QTLs for the target trait(s). GS is revolutionizing breeding practice for complex trait in domestic animals. The same approach and concepts can be readily applied to forest tree breeding. Trees also have long generation times and late expressing traits. Differently from association genetics that aims at dissecting complex traits in their discrete components, GS precludes the discovery of individual marker-trait associations and focuses on prediction of performance. By capturing the "missing heritability" of complex quantitative traits beyond the few effect variants that association genetics has so far typically identified, GS might soon cause a paradigm shift in forest tree breeding. In a prior deterministic study we assessed the impact of linkage disequilibrium (modeled by Ne and inter-marker distance), the size of the training set, trait heritability and the number of QTL on the predicted accuracy of GS . ResultsE. grandis x E. urophylla hybrids clones therefore having an effective population size of Ne= 11. The second population was a progeny trial installed by FIBRIA (FIB) with 232 full-sib families involving 120 elite parents, the vast majority F1 hybrids of E. grandis, E. urophylla, E. globulus and E. maidenii. For the GS study however only the most phenotypically relevant 75 families were used derived from intercrossing 55 parents therefore limiting the effective population size to Ne= 55. A sample of trees was taken in a stratified way from all families to provide a balanced representation of all families with similar numbers of individuals per family. Bark or leaf samples were collected, DNA extracted and genotypes obtained for 783 and 920 trees from CEN and FIB respectively. Genotyping was carried out using a high-throughput genotyping platform developed earlier [Puccinia rust resistance were obtained. Single marker regression association analyses were initially performed with all the traits, treating the markers as fixed effects. Markers selected in the previous association analysis had their effects estimated adjusting all the allelic effects simultaneously using Random Regression Best Linear Unbiased Predictor. Each population was then divided in an training set comprising 90% of the total number of individuals and a validation set with the other 10%. The estimated effects of the markers were validated using a cross validation approach using random sub-sampling with ten replications so that all individuals had their phenotypes predicted by GS. Details of the overall analytical approach have been described [This study was carried out in two structured populations of Eucalyptus represented by two progeny trials of independent breeding programs. The first was a progeny trial developed by CENIBRA (CEN) with 43 full-sib families generated by an incomplete diallel mating design carried out by intercrossing 11 highly selected Eucalyptus tree breeding practice.To date results have been obtained for height, DBH and wood density while the remaining traits are under analysis. Realized accuracies by Genomic Estimated Breeding Values (GEBV) for H and DBH were 0.67 and 0.69 for CEN and 0.62 and 0.54 for FIB while for WD in FIB it was 0.53. These accuracies match or surpass those obtained by conventional BLUP (Best Linear Unbiased Prediction) based phenotypic selection. Not surprising GEBV accuracies were low (~0.18) across populations implying variable genotype-phenotype associations across backgrounds so that population-specific GS models will be necessary. GS-based reduction in breeding time by 50%, i.e. by reducing a breeding generation time from 12 to 6 years should provide gains ≥100% in selection efficiencies. If a reduction from 12 to 3 years using flower induction can be achieved, gains in selection efficiency theoretically could surpass 300%. These are among the first experimental results of GS in forest trees and in plants in general. With the technological advances and declining costs of genotyping methods we anticipate that GS will soon be implemented operationally and revolutionize


For health professions to evolve in a highly evidence-based world it seems imperative for clinicians to build their research skills. This observational paper aims to report the research skill levels of statewide public-sector podiatrists at two different time points twelve-months apart.The Research Capacity & Culture (RCC) survey was distributed to all Queensland Health podiatrists in January 2011 (n=58) and January 2012 (n=60). The RCC is a validated tool designed to measure indicators of research skill in health professionals. Participants rate skill levels against each individual, team and organisation statement on a 10-point scale . Chi-squared and Mann Whitney U tests were used to determine any differences between survey samples.Thirty-seven (64%) podiatrists responded to the 2011 survey and 33 (55%) the 2012 survey. The 2011 survey respondents reported low skill levels (Median<4) on most individual research aspects. However, most reported their organisations’ skills to perform and support research at higher levels (Median>6). The 2012 survey respondents reported significantly higher skill levels compared to the 2011 survey in individuals’ ability to secure research funding, submit ethics applications and provide research advice (p<0.05).This study reports the research skill levels of the largest podiatry populations to date. The 2011 findings indicated that podiatrists have similarly low research skill levels to those reported in the generic allied health literature. The 2012 findings suggest podiatrists perceived higher skills and support to initiate research than in 2011. This improvement coincided with podiatry research capacity building strategies.


Mammalian artificial chromosomes are natural chromosome-based vectors that may carry a vast amount of genetic material in terms of both size and number. They are reasonably stable and segregate well in both mitosis and meiosis. A platform artificial chromosome expression system (ACEs) was earlier described with multiple loading sites for a modified lambda-integrase enzyme. It has been shown that this ACEs is suitable for high-level industrial protein production and the treatment of a mouse model for a devastating human disorder, Krabbe’s disease. ACEs-treated mutant mice carrying a therapeutic gene lived more than four times longer than untreated counterparts. This novel gene therapy method is called combined mammalian artificial chromosome-stem cell therapy. At present, this method suffers from the limitation that a new selection marker gene should be present for each therapeutic gene loaded onto the ACEs. Complex diseases require the cooperative action of several genes for treatment, but only a limited number of selection marker genes are available and there is also a risk of serious side-effects caused by the unwanted expression of these marker genes in mammalian cells, organs and organisms. We describe here a novel method to load multiple genes onto the ACEs by using only two selectable marker genes. These markers may be removed from the ACEs before therapeutic application. This novel technology could revolutionize gene therapeutic applications targeting the treatment of complex disorders and cancers. It could also speed up cell therapy by allowing researchers to engineer a chromosome with a predetermined set of genetic factors to differentiate adult stem cells, embryonic stem cells and induced pluripotent stem (iPS) cells into cell types of therapeutic value. It is also a suitable tool for the investigation of complex biochemical pathways in basic science by producing an ACEs with several genes from a signal transduction pathway of interest. Gene therapy may be extremely promising for the treatment of various genetic and acquired disorders in the near future in vivo telomere-associated fragmentation of existing chromosomes Four different strategies have been developed for the construction of artificial chromosomes The incorporation of different integrase recognition sequences and integrase enzyme genes into artificial chromosomes greatly improved the feasibility of integrating a transgene into the artificial chromosome Recently an artificial chromosome expression system (ACEs) was constructed which carry multiple copies (>50) of recombination acceptor sites (attB) for multiple unidirectional loading of expression cassettes by a modified λ integrase The recently developed novel method, in which the ACEs was combined with stem cell therapy for the treatment of a mouse model for a devastating human disorder, Krabbe’s disease www.calyxbio.com). The pATVMin vector was previously used to load transgenes onto Platform ACEs as shown on Herpes simplex virus thymidine kinase (HSVTK) expression cassette, flanked by a LoxP site downstream from the polyA signal. The LoxP site carrying pATVMin plasmid was digested with XbaI. The thymidine kinase expression cassette with the LoxP site was removed from the pmCCKO14 plasmid as an XbaI-SpeI fragment. This fragment was cloned into the LoxP site-containing, XbaI enzyme-digested pATVMin vector. The resulting plasmid was checked by DNA sequencing. The plasmid with the correct sequence was named pST (The pSEV1R (shuttle expression vector-1R) entry vector  and the amed pST .www.calyxbio.com) .The pSTRFP plasmid was constructed by cloning the coding sequence of the mCherry gene into the pST plasmid vector  as follows: the pmR-mCherry plasmid was digested with the BamHI and HindIII. The mCherry CDS  containing DNA fragment was isolated. The pSEV2 (shuttle expression vector-2) entry vector was constructed and kindly donated by Chromos Molecular Systems Inc.  . This vebio.com) . The RFPbio.com) . The pSTbio.com) . The Lacbio.com) . The pCrThe Platform ACEs-carrying Y2913D-SFS Chinese hamster ovary (CHO DG44) cell line from Chromos Molecular Systems Inc. was cultured in MEM alpha , 5% FCS, streptomycin-penicillin  and 10 mg/ml puromycin . The 1D9-16, RFPG18 and RFPG18-12 cell lines were generated in our laboratory and their properties are described in detail in the Results and Discussion section. These cell lines were cultured in the same medium as described above except that the puromycin was omitted. The 1D9-16 and RFPG18-12 cell lines were cultured in the presence of 400 µg/ml G418 . The RFPG18 cell line was cultured in the presence of 10 µM ganciclovir .Plasmid transfection experiments were performed with the Superfect reagent  as described by the manufacturer.in situ hybridization (FISH) experiments were performed with a standard protocol. The pPur  plasmid was labeled with FITC fluorescent dye and used as a probe for ACE detection. We used the Roche DIG Nick Translation Mix (1745816) or the Roche Biotin Nick Translation Mix (1745824) for labeling DNA probes.Fluorescence 5′-ACCCCCTTGCGCTAATGCTCTGTTA and NeoR1 5′-TCGATGAATCCAGAAAAGCGGCCA. The expected product size is 784 bp.Genomic DNA samples were isolated with the Zymo Research Quick-gDNA MiniPrep (Cat. No.: D3025). Site-specific integration of transgenes into the ACE chromosome was detected by PCR experiments with the following primers: 193AF: All pictures were photographed under a Zeiss Axiovision Z1 fluorescent microscope by using the Axiovision software purchased with the microscope.therapeutic Artificial Chromosomes (tACs). Although it harbors about 50 to 200 loading sites for gene delivery, the number of useful genes that can be loaded onto the presently available Platform ACEs is limited to the selectable marker genes that are available and that number is very small. In many cases, this can be a problem, as the loading of multiple genes onto the ACE chromosome might be important . Moreover, some of the selectable marker genes may have unwanted side-effects when they are expressed in elevated levels in certain tissues and organs in the body. This restricts the therapeutic and scientific applications of the Platform ACEs.A Platform ACEs chromosome with multiple recombination recognition sites for a special lambda-integrase (ACE integrase) was earlier constructed and a vector system was additionally established to deliver “useful genes” onto the Platform ACEs We report here the development of a new vector system, which makes use of only one selectable marker gene cassette for the delivery of a transgene . SubsequDiscosoma sp.) as the first gene to load onto the Platform ACEs and constructed the pSTRFP superloading vector . Twenty five cell lines had proper targeting into the ACEs without random integration (36%) and all of these cell lines expressed the RFP protein. Based on strong RFP signal and intact ACEs, we have chosen the 1D9-16 cell line for further experiments.Cre recombinase was transiently expressed in the 1D9-16 cell line to remove the neomycin-thymidine kinase selectable marker gene cassette flanked by direct LoxP sites from the ACEs chromosomes. Forty eight subclones were obtained with 10 µM ganciclovir selection. Twenty two cell lines (45%) which remained G418 resistant were discarded. The cell lines from which the neomycin-thymidine kinase selectable marker gene cassette was successfully removed were G418 sensitive and ganciclovir resistant. Twenty two cell lines (45%) carried intact ACEs and expressed the RFP protein. Based on strong RFP signal and intact ACEs, we have chosen the RFPG18 cell line for further experiments. The ACEs in the RFPG18 cell line was targeted with the pSTLZ vector (second loading of the ACEs). The LacZ gene was loaded onto the ACEs that already carried the RFP gene from the previous loading experiment. Seventy four G418 resistant cell lines were obtained. Thirty seven cell lines were positive for both RFP signal and LacZ staining (50%). Twenty one cell lines carried the LacZ and RFP genes exclusively on the ACEs (56.7%).We identified cell lines with intact ACE chromosomes carrying and functionally expressing both mCherry and LacZ genes correctly targeted onto the artificial chromosome . In thes


Management of liver injury is challenging particularly for the advanced grades. Increased utility of nonoperative management strategies increases the risk of developing massive liver necrosis (MLN). We reported a case of a 19-year-old male who presented with a history of motor vehicle crash. Abdominal computerized tomography (CT) scan revealed large liver laceration (Grade 4) with blush and moderate free hemoperitoneum in 3 quadrants. Patient was managed nonoperatively by angioembolization. Two anomalies in hepatic arteries origin were reported and both vessels were selectively cannulated and bilateral gel foam embolization was achieved successfully. The patient developed MLN which was successfully treated conservatively. The follow-up CT showed progressive resolution of necrotic areas with fluid replacement and showed remarkable regeneration of liver tissues. We assume that patients with high-grade liver injuries could be managed successfully with a carefully designed protocol. Special attention should be given to the potential major associated complications. A tailored multidisciplinary approach to manage the subsequent complications would represent the best recommended strategy for favorable outcomes. Trauma is a major contributor to death in the first four decades of life . AbdomenTransarterial embolization (TAE) has been introduced as a useful and effective tool for the management of hemodynamically stable patients with blunt hepatic trauma in the multimodality approach . The widA 19-year-old male presented with a history of motor vehicle crash. The unrestrained driver sustained mild head and chest injuries (right first rib fracture and occult pneumothorax). Upon physical examination, he was fully conscious with a Glasgow Coma Scale (GCS) of 15 and normotensive but tachycardic. Abdominal examination revealed mild generalized distension and diffuse tenderness mainly at the right upper quadrant with normal bowel sounds, digital rectal examination, and clear urine.Initial laboratory examination showed a white blood cell (WBC) count of 16000 (Ul), Lactic Acid of 2.33 (mmol/L), Alanine transaminase (ALT) of 767 (U/L), and serum glucose of 8.29 (mmol/L). The focused assessment sonography for trauma (FAST) was found positive, whereas the chest and pelvic X-rays and head CT were normal. Chest CT revealed bilateral lung contusion with minimal right sided pneumothorax and fracture of first and fifth ribs. Abdominal CT scan revealed large liver contusion and laceration (Grade 4) with blush and moderate free haemoperitonium (3 quadrants) . PatientA follow-up abdomen CT (48 hrs) revealed left sided pleural effusion and massive liver necrosis (MLN) with significant increase of peritoneal fluid along the left paracolic and pelvic compartments .Another CT after 4 weeks of TAE revealed that both lobes of the liver had diffuse nonenhancing and nonhomogenous areas replacing almost the left lobe and segments V and VIII of the right lobe with fluid loculation but no free fluid . OutpatiThe management of liver injury is challenging particularly for severe grades (Grades III–V) . Though Major liver devascularization (MLN) is a very peculiar entity of great interest. It is related to the disruption of vascular inflow to a liver portion either by the original trauma or following angioembolization . MLN hasTo the best of our knowledge, there are only few reported cases of MLN complicating TAE which are treated nonoperatively. The current report represents a case of severe liver injury (Grade IV with blush) treated with nonoperative management using TAE and complicated by MLN. The patient developed major liver devascularization to some extent related to his vascular anomalies with TAE being less selective. The pattern of liver enzymes, WBC count, clinical presentation, and subsequent scanning confirmed the diagnosis. Nevertheless, the patient managed to tolerate liver devascularization and he showed a smooth recovery without developing severe complications such as hepatic failure, sepsis, abdominal compartment, late bleeding, or multiorgan derangements. Follow-up CT scans demonstrated a great regeneration and full function, recovered liver.In conclusion, we assume that patients with high-grade severe liver injuries could be managed carefully according to a clear protocol. Special attention should be given to the major associated morbidities. A selective or rather tailored multidisciplinary approach to manage the morbid complications would represent the best recommended strategy for success and better outcomes in severe liver injury patients. Further studies are needed to support our assumption.


Polyglutamine (polyQ) diseases are genetically inherited neurodegenerative disorders. They are caused by mutations that result in polyQ expansions of particular proteins. Mutant proteins form intranuclear aggregates, induce cytotoxicity and cause neuronal cell death. Protein interaction data suggest that polyQ regions modulate interactions between coiled-coil (CC) domains. In the case of the polyQ disease spinocerebellar ataxia type-1 (SCA1), interacting proteins with CC domains further enhance aggregation and toxicity of mutant ataxin-1 (ATXN1). Here, we suggest that CC partners interacting with the polyQ region of a mutant protein, increase its aggregation while partners that interact with a different region reduce the formation of aggregates. Computational analysis of genetic screens revealed that CC-rich proteins are highly enriched among genes that enhance pathogenicity of polyQ proteins, supporting our hypothesis. We therefore suggest that blocking interactions between mutant polyQ proteins and their CC partners might constitute a promising preventive strategy against neurodegeneration. Polyglutamine (polyQ) diseases are fatal neurological disorders that affect the central nervous system. Nine known disorders belong to this family, which includes Huntington's disease (HD) and spinocerebellar ataxia type-1  by expanding a neighboring CC upon interaction with a CC from a partner protein ; 15. SucA very interesting hypothesis follows immediately from the previous model of polyQ action. It has been shown that proteins with expanded polyQ domains can form β-sheet aggregates We recently performed a cell-based screen for the identification of proteins that influence the aggregation and proteotoxicity of a toxic construct of polyQ ATXN1 , reports a set of aggregation enhancers without enrichment in CC  CC proteins that interact with a wild-type polyQ protein also interact with the corresponding pathogenic polyQ-extended protein, and (ii) these CC protein partners promote the aggregation of the pathogenic polyQ-extended protein. A careful analysis of these interactions should help us to develop therapeutic strategies that might prevent neurodegeneration in humans. We suggest that similar aggregation mechanisms might be relevant for other non-polyQ neurodegenerative diseases involving protein aggregation, such as Alzheimer's or Parkinson's disease.


The purpose of the study is to evaluate a fully automated motion corrected first pass myocardial perfusion (FPMP) MRI with semi quantitative perfusion parameter maps in patients with suspected ischemic heart disease.Coronary heart disease is the leading cause of death and disability in the US. FPMP MRI is increasingly used to assess ischemic heart disease; however respiratory motion is one of the major problems for myocardial blood flow quantification. An algorithm for motion correction, surface coil correction, temporal denoising and robust pixel-wise parameter map generation model was previously desribed [Xue H et al MICCAI 2009).This work evaluates automated workflow in a clinical setting to diagnose ischemic heart disease comparing free breathing and motion correcteted images and corresponding pixel-wise parameter map.Stress and rest FPMP images were acquired using a 1.5T scanner  in 39 patients with suspected ischemic heart disease. Short axis slices were acquired during infusion of 0.075 mMol/kg of Gadolinium  and adenosine  infusion  was administrated to induce stress. Free breathing, motion-corrected images and corresponding perfusion maps were assessed by 2 radiologists independently using the AHA 16 model and evaluated using a four point Likert scale (poor to excellent) to evaluate image quality and confidence level in presence or absence of hypo-perfusion regions. Upslope index of both free breathing and motion corrected images during stress and rest were manually calculated in non-ischemic and ischemic areas and compared to the corresponding pixel-wise parameter map generated based on motion corrected images. FPMP MRI results were subsequently compared to coronary angiogram, stress echocardiography, or SPECT.Perfusion defects were detected in 25 patients  MRI with semi quantitative perfusion parameter maps showed comparable accuracy for detection of significant coronary artery disease in patients with ischemic disease.Astellas Pharma Global Development, Inc.


The pre-Bötzinger complex (pre-BötC) is the essential core component of the brainstem respiratory central pattern generator (rCPG) in mammals. Phasic excitatory and inhibitory synaptic inputs during the respiratory cycle are thought to dynamically shape membrane potential trajectories and spiking patterns of pre-BötC neurons. These synaptic inputs reflect the functionally interacting neuron populations involved in rhythm and inspiratory-expiratory pattern generation. The dynamic patterns of these synaptic inputs have not been systematically studied and characterized experimentally. Accurate temporal reconstruction of the synaptic input patterns is important for testing current models of the rCPG that incorporate specific excitatory and inhibitory circuit mechanisms for rhythm and pattern generation. A recent computational model of the rCPG generating a normal “three-phase” respiratory pattern  specifiein situ perfused brainstem-spinal cord preparations of mature rats, which generate a three-phase respiratory pattern and provide mechanically stable conditions for intracellular recordings in functionally intact brainstem circuits.We developed analytical techniques that allow quantitative reconstruction of the patterns of synaptic conductances at high temporal resolution from intracellular recordings of membrane potential trajectories. Our approach extends previously proposed methods for extracting dynamic patterns of synaptic input conductances ,3 and prWe distinguished different types of inspiratory and expiratory pre-BötC neurons and analyzed the dynamical changes of excitatory (Ge) and inhibitory (Gi) conductances. Inspiratory neurons exhibited strong excitatory inputs (large dynamic increases in Ge) during their spiking phase followed by a wave(s) of inhibitory inputs during the expiratory period with large increases in Gi and temporal patterns consistent with two populations of inhibitory neurons providing inputs that coordinate inspiratory to expiratory and expiratory to inspiratory phase transitions. Expiratory neurons exhibited strong inhibition during the inspiratory phase, consistent with the rhythmic alternation of inspiration and expiration, and changes in Ge indicating reciprocal interactions between two major populations of inhibitory neurons coordinating the generation of two E1 and E2) phases of expiration. The reconstructed patterns of Ge and Gi are generally consistent with previously proposed circuit architecture [ and E2 p


There is no reliable predictor on arrival at hospital for neurological outcome of the patient with out-of-hospital cardiac arrest (OHCA). We hypothesize that ammonia and lactate may predict neurological outcome.We performed a prospective observational study. Non-traumatic OHCA patients who gained sustained return of spontaneous circulation and were admitted to an acute care unit were included. Blood ammonia and lactate levels were measured on arrival at hospital. The patients were classified into two groups: favorable outcome group  1 to 2 at 6-month follow-up), and poor outcome group (CPC 3 to 5). Basal characteristics obtained from the Utstein template and biomarker levels were compared between these two outcome groups. Independent predictors were selected from all candidates using logistic regression analysis.n = 10) were significantly lower than those in the poor outcome group (n = 88) . On receiver operating characteristic analysis, the optimal cut-off value for predicting favorable outcome was determined as 170 μg/dl ammonia, 12.0 mmol/l lactate . Logistic regression analysis identified ammonia (≤170 μg/dl), therapeutic hypothermia and witnessed by emergency medical service personnel as independent predictors of favorable outcome. When both these biomarker levels were over threshold, the positive predictive value (PPV) for poor outcome was calculated as 100%.Ninety-eight patients were included. Ammonia and lactate levels in the favorable outcome group (Ammonia and lactate blood levels on arrival are independent neurological prognostic factors for OHCA. The PPV with the combination of these biomarkers predicting poor outcome is high enough to be useful in clinical settings.


The twin-arginine translocation (Tat) protein export system enables the transport of fully folded proteins across a membrane. This system is composed of two integral membrane proteins belonging to TatA and TatC protein families and in some systems a third component, TatB, a homolog of TatA. TatC participates in substrate protein recognition through its interaction with a twin arginine leader peptide sequence.tatC loci and half of these classes possessed only tatC and tatA genes. Phylogenetic analysis of diverse TatC homologues showed that they were primarily inherited but identified a small subset of taxonomically unrelated bacteria that exhibited evidence supporting lateral gene transfer within an ecological niche. Examination of bacilli tatCd/tatCy isoform operons identified a number of known and potentially new Tat substrate genes based on their frequent association to tatC loci. Evolutionary analysis of these Bacilli isoforms determined that TatCy was the progenitor of TatCd. A bacterial TatC consensus sequence was determined and highlighted conserved and variable regions within a three dimensional model of the Escherichia coli TatC protein. Comparative analysis between the TatC consensus sequence and Bacilli TatCd/y isoform consensus sequences revealed unique sites that may contribute to isoform substrate specificity or make TatA specific contacts. Synonymous to non-synonymous nucleotide substitution analyses of bacterial tatC homologues determined that tatC sequence variation differs dramatically between various classes and suggests TatC specialization in these species.The aim of this study was to explore TatC diversity, evolution and sequence conservation in bacteria to identify how TatC is evolving and diversifying in various bacterial phyla. Surveying bacterial genomes revealed that 77% of all species possess one or more TatC proteins appear to be diversifying within particular bacterial classes and its specialization may be driven by the substrates it transports and the environment of its host. As of June 2012, the number of available completed bacterial genome sequences was 4373 (2422 are plasmids and 1951 are chromosomes) and corresponded to a total of 1018 bacterial species. tBLASTn searches resulted in a total of 1415 genomic tatC loci out of a total 782 bacterial species . A list of all TatC sequences identified in this study is provided in tatC sequences, a summary of Tat translocase components  identified in tat operons for each bacterial class surveyed in this study is also provided and listed in Ilyobacter polytropus, from the order Fusobacteriales, which had a plasmid encoded tatC.The TatC protein and nucleotide sequence datasets analyzed in this study were collected from completed sequences bacterial genome collection on the National Center of Biotechnology Information (NCBI) website (Multiple Sequence Comparison by Log-Expectation (MUSCLE), Multiple Alignment using Fast Fourier Transform (MAFFT) and hidden markov model alignment  software of the HMMER3 v3.1 package http://pfam.sanger.ac.uk/) with accession PF00902. The application of the hmmalign software aided in the multiple alignment of distantly related TatC homologue sequences. Non-confidently aligned residue columns were omitted from phylogenetic analyses and synonymous/non-synonymous nucleotide substitution analyses. Poorly aligned residues within amino- and carboxyl- termini and within loop regions were generally removed from multiple alignments due to low conservation in these regions.Multiple sequence alignments of TatC amino acid sequence datasets used for phylogenetic and synonymous/non-synonymous analyses and consensus sequence determinations were performed using three different programs, http://toolkit.tuebingen.mpg.de/blastclust) to aid in the selection of 232 unique, taxonomically diverse TatC sequences that represented all bacterial phyla surveyed. Multiple alignment editing was performed with custom Python scripts and with the multiple alignment editing program Jalview To selectively represent non-redundant taxonomically diverse TatC sequences for phylogenetic and synonymous/non-synonymous analyses the 1415 TatC dataset was analyzed using the web based BLASTClust program (transmembrane hidden markov model (TMHMM) dense alignment surface (DAS) membrane protein structure and topology (MEMSAT) TatC transmembrane domain predictions for TatC sequences were performed using local installations of programs Vulcanisaeta distributa TatC protein sequence (YP_003902595.1) served as the outgroup for phylogenetic analysis (E. coli W strain TatC (YP_006175467.1) was the outgroup for Bacilli TatC analysis  adjacent to the tatC locus/loci from annotated tatC sequence locus tags during genome surveys described above. In some cases, tatA and tatB loci were also sought out within these genomes to confirm legitimate absences from the TatC locus using NCBI tBLASTn searches with E. coli TatA (GenBank: YP_006126698) and TatB (GenBank: YP_006126699) as query sequences. The results of this analysis are presented in Bacterial tatC loci present within Bacilli genomes  was determined by the equation [Freq = (ngene/nlocus ) x OtatC] where ngene is the number of times a gene is identified at one of ten loci before or after the tatC locus, nlocus is the number of total loci examined, and OtatC is total number of tatC operons examined. The reading frame of all genes was also considered in the frequency of gene occurrence calculations as well as its proximity to the tatC locus. All of these parameters were used to generate the tatC operon diagrams shown in the right hand panel of Examination of synonymous non-synonymous analysis program (SNAP) package http://hcv.lanl.gov/content/sequence/SNAP/perlsnap.html). Generation of codon aligned tatC nucleotide datasets used for this analysis were performed with the program RevTrans tatC sequences are summarized in tatC sequence included in the analysis, a mean dS/dN value was calculated from all pairwise comparisons to other sequences in the same selected dataset. Pairwise dS/dN values could only be determined for 39% of all pairwise alignments of 232 tatC sequences included in this study since the remaining pairwise dS/dN values had pS values of 0.75 that prevented a reliable Jukes-Cantor transformation calculation of the dS value. Mean dS/dN values are reported in boxplot function as part of the R program version 2.15.2 (http://developer.r-project.org/). The number of observed synonymous nucleotide substitutions (Sd) at each codon was calculated using the Perl script codons-xyplot.pl available within the SNAP software package and the mean Sd values are summarized in tatCd and tatCy were also analyzed using SNAP to determine pairwise dS/dN values for the entire protein in addition to Sd values for each codon in the multiple alignment resulting in mean Sd values.Synonymous to non-synonymous nucleotide substitution analyses were performed with E. coli TatC structure was generated from the recently published X-ray crystal structure of Aquifex aeolicus TatC E. coli TatC (GenBank: YP_006175467.1) and A. aeolicus TatC (Swiss-Prot: O67305) protein sequence. This alignment was used to generate 10 models based on the A. aeolicus TatC crystal structure  of species with tatC homologues were typically facultative anaerobes, obligate anaerobes and facultative aerobes. Very few species possessing Tat systems had an obligate aerobic physiology (∼11%) in this survey. This indicates that the Tat system is linked to bacteria that rely upon an anaerobic physiology. However, this finding also supports experiments in E. coli that show a constitutive expression of tat genes under both aerobic and anaerobic conditions Before beginning phylogenetic and sequence-based analyses of bacterial is study . Surprisnt locus . This matatC homologue representing either Gram- negative or Gram- positive bacteria had reduced genome sizes ≤2 Mb and/or were obligate and opportunistic parasites which confirm findings from previous analysis tatC homologues within Gram- negative bacterial genomes appeared to be related to the size of the genome, as genomes ≤2 Mb typically lacked tatC sequences. For example, the obligate parasite phylum Mollicutes  lacked tatC homologues as well as members from the thermophilic Thermotogae phylum . An exception was observed for obligate endosymbiotic α-proteobacterial members from Rickettsiales, with small average genome sizes of ≤1.3 Mb, yet all possessed a tatC homologue. Since many species within Rickettsiales are also known to be free-living, such as Pelagibacter ubique (SAR11), there may be a greater requirement for the Tat system in this expanding bacterial class. Gram- positive species that lacked recognizable tatC loci were generally opportunistic microflora of plants and animals, typically pathogens, likely due to the genome sequencing bias favouring pathogenic organisms. For instance, Bifidobacteriaceae of the phylum Actinobacteria, was the only Actinobacterial subgroup that lacked tatC homologues with the exception of Bifidobacterium longum.Overall, almost all species that lacked a tatC locus per genome was relatively narrow, where only 39 species from diverse phyla  had at least two tatC loci as listed in tatC homologues was limited to Bacilli species, namely Bacillus subtilis and Bacillus halodurans, that are known to encode for two TatC isoforms, TatCd and TatCy. This tatC survey identified additional species with multiple tatC isoforms in the Bacilli class, Bacillus, in particular within Paenibacillus, Lysinibacillus and Geobacillus genera  and those in possession of more than one tatC locus typically shared a common environmental habitat (in particular those organisms living in soil and/or sea). Like Bacilli, the presence of multiple tatC loci/genome suggests that one or both of these additional Tat systems may support a specialized secretion function or alternatively protein secretion under a restrictive physiology as observed for Bacilli TatC isoforms. Hence, the presence of an additional Tat translocase system may allow a competitive advantage by supporting a specific protein secretion compared to other bacteria that possess a single TatC system in the same ecological niche.The distribution of bacteria in possession of more than one s genera . In facttatC loci and that only 5% have ≥2 tatC loci  was performed to expand upon previous studies involving smaller datasets (∼70 sequences) tatC members within bacteria, and to determine the origin of Bacilli TatC isoforms TatCd and TatCy. A chrenarchaeal Vulcanisaeta distributa TatC protein sequence served as the outgroup for this phylogenetic analysis as it represents one of the most ancient microorganisms that possesses a TatC homologue.Phylogenetic analysis of 232 TatC proteins representing diverse taxa from all bacterial classes in possession of one or more tatC and other genes as their genome reduced during evolution tatC genes in the genomes of other obligate intracellular parasites observed in our TatC distribution survey strongly supports this argument. Clades 1–3 also demonstrated evidence supporting internal gene duplication events, as many species in possession of more than one TatC in these clades branched together at the same nodes . This indicates that horizontal gene transfer influences clade 4 branching patterns in contradiction to previous findings Branching arrangements within the fourth clade at node 4 demonstrated a different clustering of TatC homologues from diverse taxonomic origins and included species from Actinobacteria, Cyanobacteria, Deinococcus-Thermus, Acidobacteria phyla, and from the class δ-proteobacteria . The lactatC loci in the genomes of eight species dispersed throughout the phylogenetic tree from γ-proteobacteria (Colwellia psychrerythraea), δ-proteobacteria (Geobacter lovleyi), Actinobacteria , Bacteroidetes (Cytophaga hutchinsonii), Acidobacteres (Candidatus Solibacter usitatus) and Nitrospira (Candidatus Nitrospira defluvii). Each homologous TatC pair from these 8 organisms grouped independently with PP≥0.9 supporting gene duplication events .Internal gene duplication events may explain the presence of multiple n events . NonethePhylogenetic analysis of the TatC protein family demonstrated evidence for combination of linear and environmental-driven inheritance, suggesting that evolutionary pressures acting upon TatC in free-living organisms could account for specialized Tat systems able to confer evolutionary advantages in environmental niches with strong ecological competition. The occurrence of multiple TatC homologues in single species apparently arisen from either intra-genomic events of gene duplication or horizontal gene transfer events further supports this hypothesis, thus potentially expanding the idea of the presence of functionally distinct Tat systems within single organisms, so far narrowed to Bacilli class tatC loci found in Bacilli genomes appeared to be evolving separately from all other multiple TatC homologues,  and integrases (xerC) were identified in a 8 gene radius in either frame from tatC loci as shown during operon conservation analysis  with genes associated with molybdenum cofactor biosynthesis proteins , with chaperones (groE), and 40% frequency of association to various vitamin/cytochrome and cofactor biosynthesis genes  transporters and cofactor biosynthesis, such as high-affinity Fe2+/Pb2+ permease (FTR1/efeU), iron binding lipoprotein/imelysin peptidase M75 (COG2822) and the known Tat substrate ferrous iron dependent DyP-type peroxidase (efeB/ywdH) tatC operons examined in these nodes. Previous experiments have shown that the secretion efeB is effected by the environmental salinity of the growth medium tatCy operon at nodes y5–y7, such as thioredoxin reductases (trx/ydbP) E. coli tatC operons, specifically ubiquinone oxidoreductases and methyltransferases were also found at a frequency of 5% and 18% respectively in Bacilli tatC loci.Examination of the TatCd and TatCy clades in n region . tatCy ois genes . As TatCtatCd operon analysis at branch node d1 in this sub-branch showed high gene associations to the Tat substrate phosphodiesterase D (phoD) gene that confirms previous findings pcp), a periplasmic protein involved in pyroglutamyl signal sequence cleavage B. subtilis TatCd dependent process and its secretion is influenced by phosphate availability phoD gene in the tat operon and in its place had conserved genes with as yet undefined functions . Additionally, genes encoding proteins involving a bound fatty acid like DegV domains (degV) and dihydrofolate reductase (dfrA/DHFR) known to be tightly folded protein in the cytoplasm tatC operons surveyed in nodes d2–d3  in comparison to those classes with higher tatC conservation (dS/dN >1) that indicated sequences that are actively maintained in the host genome. Overall, the mean dS/dN value of all pairwise comparisons of tatC homologues was 2.62, indicating that bacterial tatC sequences are maintained under selective pressure, albeit much lower than initially expected. This result reflected a high proportion of non-synonymous sequence variation occurring in different bacterial classes and dS/dN values for species from various bacterial classes ranged from 0.35 to 5.99 (The ratio of synonymous (dS) to non-synonymous (dN) nucleotide substitution was calculated for all pairwise comparisons of gene sequences coding for 232 taxonomically representative TatC homologues. The purpose was to determine which bacterial classes possessing  to 5.99 .tatC possessing classes showed that five classes including Actinobacteria and Deinococci had the lowest median dS/dN values (1.0–2.0) in comparison to γ- proteobacteria, ε- proteobacteria, Bacteroidetes and Bacilli with the highest median dS/dN (3.0–4.0) values  values . Low medclade 4; .tatC classes examined (52% of 23 classes) resided within the intermediate median dS/dN value range (2.1–3.1) as shown in tatC homologues represented a diverse group largely composed of Gram- negative organisms and a single Gram- positive class (Clostridia). The intermediate median dS/dN values for these bacterial classes indicated that the overall mean dS/dN value of 2.62 accurately reflected the majority of bacterial class tatC sequence variation in bacteria. The broadest ranges of dS/dN values among bacterial classes in this group were found in within Verrucomicrobia (2.74–3.76), β-proteobacteria (1.62–5.10) and δ- proteobacteria (2.10–3.36), strongly suggesting that tatC homologues in these classes have significantly different selective pressures directing sequence conservation. High tatC dS/dN values observed for β-proteobacteria and δ-proteobacteria were particularly interesting, since high dS/dN values generally corresponded to species that possessed more than one tatC locus/genome  were related to tatC homologues from Methylobacillus flagellatus and Methylotenera mobilis, suggesting that multiple tatC copies may have greater pressures to maintain synonymous conservation to support conditionally dependent Tat system substrate secretion activity in these methylotrophic organisms.The majority of bacterial s/genome . For exatatC homologues in these classes were maintained under the highest selection pressure. Bacterial classes with high median tatC dS/dN values also tended to possess more than one tatC locus/genome, as observed for multiple tatC sequences found in species from Bacilli and γ- proteobacteria  for Bacilli and γ- proteobacterial species with more than one tatC copy suggesting that multiple tatC copies have greater selective pressures acting upon them. As dS/dN values increased in bacterial classes we surveyed, the frequency of identifying multiple tatC copies also increased  proved to be an exception to this trend and supports a role for lateral gene exchange in common niches influencing high dS/dN values.The highest median dS/dN values (>3.1) were observed for γ- proteobacteria, ε- proteobacteria, Bacteroidetes, and Bacilli and indicated that bacteria . As obsencreased . HowevertatC homologues from various classes, revealed that the selective pressures acting upon tatC sequence variation support our phylogenetic analyses, which demonstrates that TatC evolution is largely directed by the taxonomic origins of its host but also appears to be driven by its environmental pressure. Multiple tatC loci present in bacterial classes also tend to be maintained at higher dS/dN (with the notable exception of Actinobacteria), suggesting that tatC duplicates fulfill important functional purposes for their host.Examination of dS/dN analysis of bacterial Analysis of individual TatC amino acid and nucleotide conservation from multiple alignments was performed to identify any conserved residues within TatC and identify other conserved residues involved in substrate binding regions and/or TatC multimerization contacts. This was carried out by calculating the extent of conservation from aligned amino acids and the extent of synonymous substitutions in each corresponding codon sequence in the nucleotide codon alignment. The most conserved amino acid according to its percentage identity (% identity) and its associated mean synonymous nucleotide substitution (Sd) provide an estimation of the selective pressure acting on particular residues and domains within the protein. The TatC protein alignment used for phylogenetic analyses and its corresponding codon aligned nucleotide sequences were used for these analyses. This resulted in an amino acid sequence where the highest % identity was reported for each residue in alignment (the consensus sequence) in addition to its respective mean Sd value for each codon.A. aeolicus TatC structure E. coli model of TatC  was shown to be important for TatC structure and function based on site-directed mutagenesis experiments (The mean synonymous substitution (Sd) values identified many regions undergoing variable rates of conservation throughout the bacterial TatC protein . Our ana of TatC . Examinaeriments  [29], [3E.coli TatC translocation function when replaced with either Ala Our analysis of TatC residue conservation identified a total of eleven highly conserved (≥75% identity) amino acid sites from TM3 to the carboxyl terminus of TatC  and poorly conserved (≤25% identity) residues were mapped onto a model structure of tat operons lack tatB, this analysis was also performed to determine residues that interacted with TatA and TatC specifically, and identify conserved residues specific for only TatA-TatC associations. A comparative TatC-TatCy/Cd conservation (according to % identity) consensus alignment is provided for each amino acid position from amino- to carboxyl- terminus in The final objective of this study was to compare the extent of amino acid conservation within Bacilli TatCd and TatCy isoforms to the bacterial TatC consensus to identify regions or residues in each TatC isoform that may contribute to specialized substrate secretion in Bacilli. Since Bacilli 91GLYK94 motif in both Bacilli TatCd (TP94) and TatCy (YE100) suggest that these residues might contribute to signal sequence specificity since these regions were shown to crosslink to the twin arginine leader peptide in E. coli experiments In general, highly conserved residues identified in the overall bacterial TatC consensus were generally similar to those identified in Bacilli TatCd and TatCy, and the majority of variation from the main consensus was observed sporadically after loop1 throughout the carboxyl terminus of the protein . NotableE. coli TatC photoaffinity crosslinking experiments tatB, our bacterial TatC alignment would be expected to see moderate conservation of TatB-TatC interacting residues  with transposon/integron gene elements compared to TatCd. This suggests that Bacilli TatCd have undergone rapid diversification from their originally duplicated TatCy sources. The combination of Bacilli TatC phylogenetic analysis with tatC operon mapping, also revealed an assortment of interesting gene associations that may be potential substrates/or linked to pathways specific for TatCd/y isoform substrates  followed taxonomic inheritance in agreement with previous findings  out of 4bstrates . This anndidates . It is atatC encoding taxa determined that tatC sequences were more variable sequence in Actinobacteria and Deinococci while γ- proteobacteria, ε- proteobacteria, Bacteroidetes and Bacilli were maintained under the highest rates of conservation  within diverse ervation . This shn system –7.Figure S1A protein alignment of all 233 TatC proteins examined in this study. Amino acids for each of the 233 TatC proteins are provided and show all alignable positions examined in this study. Abbreviated TatC sequence tags are provided for clarity and correspond to bacterial genus and species names provided in Additional file 1. Regions highlighted in blue have been predicted to be transmembrane strand regions. The degree of blue highlighting behind each amino acid indicates how many transmembrane prediction programs have assigned that amino acid to a transmembrane segment (three to one according to the shift from the darkest to the lightest blue) Yellow and black bar charts shown at the bottom x-axis of the amino acid alignment indicate the amino acid conservation (top yellow chart) where bar height is proportional to the conservation of physico-chemical properties for each column of the alignment, quality (middle yellow chart) where bar height and shading is proportional to the number of alignable residues and % amino acid identity at each position and the amino acid consensus (bottom black chart) representing the most frequently identified amino acid for each alignable position.(PDF)Click here for additional data file.Figure S2Vulcanisaeta distributa TatC sequence  served as the outgroup for this analysis. Symbols adjacent to taxa indicates multiple TatC copies from the same genomes discussed in the Results and Discussion section and the corresponding species are detailed as follows: Cytophaga hutchinsonii (•); Geobacter lovely (••); Colwellia psychrerythraea (•••); Xylanimonas cellulosilytica (x); Streptomyces hygroscopicus (xx); Candidatus Solibacter usitatus (xxx); Nakamurella multipartita (¥); Candidatus Nitrospira defluvii (¥¥); Sulfobacillus acidophilus (Sa); Thermodesulfobium narugense (Tn); Thermodesulfatator indicus (Ti). Numbering in light blue beneath each node represents posterior probability (PP) confidence estimates where 1 represents branching patterns with the highest confidence values and 0 no confidence in relationship at the node. Only PP values equal or greater than 0.6 are shown. Nodes with large black numbers highlight important clade divisions (1–4) between branches and Greek delta (d) symbols written beside these clade numbers indicate important delta proteobacterial divisions within that numbered clade. Bacterial classes enriched in major clusters are highlighted on the right hand side of the dendrogram, and in the case of Bacilli TatC sequences, TatCd and TatCy isoforms are also indicated.A rooted phylogenetic tree of bacterial TatC proteins. The rooted dendrogram was generated by Bayesian analysis of 233 TatC sequences from diverse bacterial taxa where the archaeal (PDF)Click here for additional data file.Table S1tatC genome sequences obtained from NCBI for this study.A complete list of all 1415 bacterial (XLS)Click here for additional data file.Table S2tatC sequences from various bacterial classes.The distribution of mean synonymous to non-synonymous (dS/dN) substitutions within (XLS)Click here for additional data file.


Venturia inaequalis, the pathogenic agent of apple scab, cause severe damage in apple cultivation. In commercial apple production, pesticides are employed frequently and in high quantities. Alternative methods such as the use of mycoparasitic Trichoderma fungi to control fungal plant pathogens have been explored [Trichoderma chitinases has lead to an increase of the trees’ resistance against apple scab [Trichoderma chitinase genes within the plant. At the Julius Kühn-Institute in Dresden, the apple cultivar 'Pinova' was transformed by introducing the construct pBIN (Endo + Nag) into the transgenic lines. With the help of promoter CaMV 35S the transgenic lines constitutively express endochitinase gene ech42 and exochitinase gene nag70 from the biocontrol fungus Trichoderma atroviride. In order to study whether the constitutive expression of ech42 and nag70 has an effect on mycorrhization of plants with transgenic roots  and – as apple cultivars are propagated vegetatively by grafting– whether expression of ech42 and nag70 in a transgenic scion grafted on a non-transgenic rootstock has an effect on mycorrhization (systemic effect), a series of greenhouse experiments was conducted. In a first experiment, non-grafted clones of wildtype apple cultivar ‘Pinova’ and two transgenic lines were cultivated in artificial substrate and inoculated with two AMF species, Glomus intraradices and G. mosseae. After four months of cultivation, root colonization rates were significantly lower in the transgenic lines compared to the non-transformed cultivar 'Pinova' [ech42 and nag70 in a transgenic scion grafted on a non-transgenic rootstock) on mycorrhization, two further experiments were carried out. Grafted sample trees  were cultivated in the greenhouse in pots with artificial substrate and inoculum of Glomus intraradices and G. mosseae. Root samples were taken after four and eight months. A second set of grafted sample trees was cultivated in pots with soil from an intensively managed plantation in the ‘semi-natural’ environment of a gossamer cage. Root samples were taken after 12 and 24 months. For all experiments, root colonisation rates were determined microscopically. Molecular methods – PCR with taxa specific primer sets [The project 'Mycorrhizal symbioses of transgenic apple trees with increased resistance against fungal pathogens'  investigates whether the transformation of apple trees with fungal chitinase encoding genes - besides increasing resistance against fungal pathogens - also interferes with mycorrhizal fungi. Pathogenic fungi such as the ascomycete species explored . Geneticple scab . Howeverple scab . Like inple scab . Consequmer sets , cloning


A 75-year-old long-term male smoker and poorly controlled hypertensive presented with a 3-month history of intermittent epistaxis refractory to cauterisation with silver nitrate. Nasopharyngeal examination revealed a mass in the post nasal space. An urgent endoscopic biopsy confirmed an extraosseous plasmacytoma. Post operative radiotherapy was scheduled. No evidence of recurrence of disease following completion of treatment has been detected during clinical surveillance.Epistaxis; Nasopharynx; Biopsy; Plasmacytoma; Radiotherapy A 75-year-old long-term male smoker and poorly controlled hypertensive presented with a 3-month history of intermittent epistaxis refractory to cauterisation with silver nitrate. Nasopharyngeal examination revealed a smooth mass extending inferiorly from the right fossa of Rossenmuller and effacing the posterior pharyngeal wall . An urgeSerum and urine assays for Bence Jones protein were negative and there was no evidence of gammopathy. A bone marrow biopsy and Computed Tomography and Magnetic Resonance imaging did not demonstrate any metastasis or skeletal involvement. The Excisional biopsy had been complete and the patient underwent radical radiotherapy with a 45Gy dose in 20 fractions of the nasopharyngeal field.No evidence of recurrence of disease following completion of treatment has been detected during clinical surveillance.A plasmacytoma is a very rare discrete solitary mass of neoplastic monoclonal plasma cells, first described by Schridde in 1905 . They reTissue biopsy, serum electrophoresis (to exclude myeloma) and radiological skeletal survey with bone marrow biopsy to determine skeletal involvement is necessary for diagnosis. Treatment includes a combination of surgical excision and radiotherapy. Follow-up radiological and electrophoresis assessment is required after treatment to detect recurrences and progression to myeloma (10-30% frequency). The overall 10 year survival is 70% .


A previous study by our group  showed a significant allergic gastrointestinal inflammation in birch pollen allergic patients with gastrointestinal symptoms during the birch pollen season. The pathophysiological mechanisms of this type of allergic reaction are still poorly studied. However it is not yet explored if all birch pollen patients have signs of gastrointestinal inflammation during the pollen season.To study the immune pathology of the duodenal mucosa in birch pollen allergic patients without gastrointestinal symptoms, outside and during the birch pollen season.All patients were clinically diagnosed as birch pollen allergic. Seven patients allergic to birch pollen without any gastrointestinal symptoms and seven completely healthy individuals were included in the study for comparison. All patients were examined with a standard skin prick test panel and blood samples were analysed for IgE antibodies by component micro-arrays (ISAC). The two groups of patients underwent gastroscopy and duodenal biopsies were taken both during (May-June) and outside the pollen season (November-January) for each patient. Duodenal biopsies were examined with immunostaining for mast cells (IgE and tryptase), eosinophils (eosinophil peroxidase), T-cells (CD3), and dendritic cells (DC) (CD11c).In the pollen allergic group there was a significant increase in eosinophils, mast cells and DC’s but not T cells during the pollen season as compared with values outside the season. Interestingly, in the healthy control group we observed a significant increase in CD3+ cells during the birch pollen season, but no change in the other cell types during the pollen season.Patients allergic to birch pollen have, regardless of subjective gastrointestinal symptoms, clear signs of an ongoing allergic inflammation in their intestinal mucosa during the pollen season.


This case report describes the use of high-resolution magnetic resonance imaging (HRMRI) to visualize basilar artery atherosclerotic plaque in a patient with a pontine stroke. HRMRI with three-dimensional image acquisition was used to visualize plaque in several planes to localize arterial wall pathology. Fluid attenuated inversion recovery (FLAIR) sequences of the basilar artery showed wall thickening throughout the basilar artery wall and good contrast between the artery wall and cerebrospinal fluid. Intracranial atherosclerosis (ICAD) is a common cause of stroke, but the pathology is not well understood because it cannot be easily studied in living patients. In extracranial carotid arteries, high-resolution magnetic resonance imaging (HRMRI) has shown good correlation with pathology , but carA 55-year-old man with atherosclerotic risk factors presented with acute onset dysarthria and left hemiplegia consistent with the classic clinical syndrome pure motor hemiplegia due to basilar artery branch occlusion . He was In this report, we demonstrate that HRMRI with 3D image acquisition can visualize intracranial plaque in several planes with good spatial resolution. Small improvements in spatial resolution are important when imaging small structures like the basilar artery. We found that an available FLAIR sequence with resolution of 0.4 mm showed good visualization of the atherosclerotic wall, likely due to the suppression of CSF signal. Unlike in T1/PD-weighted VISTA images wherein the vessel wall remained visible in normal volunteers , in our The patient's pontine infarct was proximal to his basilar stenosis A–D and tAt the stenosis , the artHRMRI with 3D image acquisition can visualize basilar artery plaque in multiple planes, allowing identification of plaque features that may contribute to the clinical presentation. The addition of FLAIR sequences helps localize arterial wall pathology by suppressing the surrounding CSF signal.