Computed tomography (CT) scanning has become essential in the early diagnostic phase of trauma care because of its high diagnostic accuracy. The introduction of multi-slice CT scanners and infrastructural improvements made total-body CT scanning technically feasible and its usage is currently becoming common practice in several trauma centers. However, literature provides limited evidence whether immediate total-body CT leads to better clinical outcome then conventional radiographic imaging supplemented with selective CT scanning in trauma patients. The aim of the REACT-2 trial is to determine the value of immediate total-body CT scanning in trauma patients.The REACT-2 trial is an international, multicenter randomized clinical trial. All participating trauma centers have a multi-slice CT scanner located in the trauma room or at the Emergency Department (ED). All adult, non-pregnant, severely injured trauma patients according to predefined criteria will be included. Patients in whom direct scanning will hamper necessary cardiopulmonary resuscitation or who require an immediate operation because of imminent death (both as judged by the trauma team leader) are excluded. Randomization will be computer assisted. The intervention group will receive a contrast-enhanced total-body CT scan (head to pelvis) during the primary survey. The control group will be evaluated according to local conventional trauma imaging protocols (based on ATLS guidelines) supplemented with selective CT scanning. Primary outcome will be in-hospital mortality. Secondary outcomes are differences in mortality and morbidity during the first year post trauma, several trauma work-up time intervals, radiation exposure, general health and quality of life at 6 and 12 months post trauma and cost-effectiveness.The REACT-2 trial is a multicenter randomized clinical trial that will provide evidence on the value of immediate total-body CT scanning during the primary survey of severely injured trauma patients. If immediate total-body CT scanning is found to be the best imaging strategy in severely injured trauma patients it could replace conventional imaging supplemented with CT in this specific group.NCT01523626).ClinicalTrials.gov: ( Injuries are the cause of 5.8 million deaths annually which accounts for almost 10% of global mortality . This bu®) guidelines which include a fast and priority-based physical examination as well as screening radiographs supplemented with selective Computed Tomography (CT) [Specialized trauma centers all over the world provide initial trauma care and diagnostic work-up of trauma patients. This work-up is standardized and frequently based on the Advanced Trauma Life Support (ATLSphy (CT) . ATLS guIn recent years CT has become faster, more detailed and more available in the acute trauma care setting. CT shows high accuracy for a wide range of injuries -7 which In the past, CT scanners were located in the radiology department, frequently even on another floor than the emergency department (ED) where the trauma patient is admitted. The past assumption that total-body CT scanning in severely injured trauma patients is too time consuming may no longer be held, since an increasing number of trauma centers have a CT scanner available at the ED or even in the trauma room itself ,12. SeveThe most important question remains whether immediate total-body CT scanning will translate to improved clinical outcome. A recent study in 4621 trauma patients reported a significant increase in the probability of survival for patient given immediate total-body CT scanning compared with conventional imaging strategies supplemented with selective CT scanning . HoweverAlthough literature provides limited evidence for the usage of an immediate total-body CT scan in the work-up of trauma patients, more and more trauma centers encourage and are implementing immediate total-body CT scanning in the diagnostic phase of primary trauma care. Since the burden of total-body CT scanning in terms of costs and radiation dose is at least controversial ,27,28, tIn order to assess the value of immediate total-body CT scanning in severely injured trauma patients, the Academic Medical Center (AMC) in Amsterdam, the Netherlands, has initiated an international multicenter randomized controlled trial. Severely injured patients, who are thought to benefit the most from a total-body imaging concept, will be included. Such a trial has never been done before and is crucial to provide evidence whether or not the usage of immediate total-body CT scanning in the diagnostic phase of primary trauma care is justifiable.®) based radiological imaging. The secondary objectives are to assess the effects of total-body CT scanning on long term clinical outcomes, quality of life, clinically relevant time intervals in the early phase of trauma care and the differences in treatment strategies used.The primary objective is to determine the effects of immediate total-body CT scanning during the primary trauma survey on clinical outcomes compared to patients who are evaluated with standard conventional Advanced Trauma Life Support  stabilize the vital functions. Usually, peripheral intravenous access is taken care of by the ambulance personnel, but if not, at least one intravenous catheter will be inserted before radiologic imaging takes place. Based on information received from the ambulance personnel and the findings during primary survey, the trauma team leader decides on the eligibility of the patient to participate in the trial. If the patient is found to be eligible randomization takes place. Figure The intervention group will receive a total-body CT scan from head to pelvis. In the intervention group conventional radiography of the torso and FAST will be completely omitted. The CT protocol for the consists of a two-step whole-body acquisition (from vertex to pubic symphysis) starting with Head and Neck Non Enhanced CT (NECT) with arms alongside the body. The preferred technique for the second complementary scan is a split-bolus intravenous contrast directly after repositioning of the arms alongside the head, and this second scan covers thorax, abdomen and pelvis. Participating centers however are free to choose their own technique as long as intravenous contrast is given for the chest and abdominal part of the total-body CT.The control group will be evaluated according to a conventional trauma protocol with selective CT scanning. The REACT-2 trial has been designed to maximize the applicability of the trial's results to usual care settings. Therefore, the technical details of the CT scanning done in the control group are not specified and participating centres follow their own protocols. Indications for the selective CT scanning however are pre-defined based on the combined local protocols of the participating centers. These standardized protocols provide a basis for the comparison of the two imaging approaches.All non-pregnant trauma patients aged 18 years and older having life-threatening  conditions with compromising vital parameters, with clinical suspicion on specific injuries or with specific injury mechanisms are included. Patients in whom the scanning will hamper necessary (cardiopulmonary) resuscitation or who require an immediate operation because of imminent death (both as judged by the trauma team leader) are excluded. Detailed in- and exclusion criteria are summarized below:Trauma patients with the presence of life-threatening vital problems defined as at least one of the following:- respiratory rate ≥ 30 min of ≤ 10/min;- pulse ≥ 120/min;- systolic blood pressure ≤ 100 mmHg;- estimated exterior blood loss ≥ 500 ml;- Glasgow Coma Score ≤ 13;- Abnormal pupillary reaction onsite.ORPatients with one of the following clinically suspicious diagnoses:- flail chest, open chest or multiple rib fractures;- severe abdominal injury;- pelvic fracture;- unstable vertebral fractures/spinal cord compression;- fractures from at least two long bones.ORPatients with one of the following injury mechanisms:- fall from height (> 3 m/> 10 ft);- ejection from the vehicle;- death occupant in same vehicle;- severely injured patient in same vehicle;- wedged or trapped chest/abdomen.Trauma patients with one of the following characteristics will be excluded:- known age < 18 years;- known pregnancy;- referred from another hospital;- clearly low-energy trauma with blunt injury mechanism;- penetrating injury in 1 body region (except gun shot wounds) as the clearly isolated injury;- any patient who is judged to be too unstable to undergo a CT scan and requires (cardiopulmonary) resuscitation or immediate operation because death is imminent according to the trauma team leader in mutual agreement with the other leading care givers.The primary outcome criterion for this trial is in-hospital mortality.As secondary parameters for the trial focus on additional clinical consequences for the patients and cost-effectiveness and cost-utility:- mortality ;- morbidity interventions and re-admissions up to 6 months post trauma; transfusion requirements, length of ICU stay and number of ventilation days);- several time intervals during initial evaluation ;- radiation exposure;- quality of life 6 and 12 months after the trauma as recorded by completing the EuroQol-6D;- general health 6 and 12 months after the trauma as recorded by completing the HUI-3;Economic parameters/endpoints:- total costs of imaging during the initial/index hospital stay;- total direct and indirect medical and non-medical costs during the first half year posttrauma;- quality adjusted life-years (QALY's).If a patient is eligible for the trial the diagnostic imaging pathway for initial assessment in the trauma resuscitation room will be determined by randomization. The randomization will be performed immediately after inclusion at computers located in the trauma room of the participating hospitals. Randomization will be performed using a 'one-click' computer program on a 1:1 basis per hospital with varying block sizes of 2, 4, 6, 8, 10 and 12. The trauma team will be directly informed on the outcome of the randomization so that imaging can be started. A standardized case record from (CRF) will be used. This CRF is totally web-based via a secured internet module.A previous study reported a reduction in mortality from 15% to 8.6% with total-body CT scanning as the single diagnostic procedure during trauma evaluation as compared to historical control data . AnalysiThe main analyses of primary and secondary outcomes will be conducted for all randomized patients according to the result of the randomization (intention-to-treat). Data are expressed as percentages for categorical data, as mean and standard deviation (SD) for normally distributed numerical data and as median, range, and, where appropriate, inter-quartile range (IQR = 25 to 75%) for non-normally distributed numerical data.The following subgroups will be used for subgroup analysis:- multitrauma patients (defined as Injury Severity Score (ISS) >/=16);- severe traumatic brain injury patients  ≤ 8 and an Abbreviated Injury Score (AIS)-head of ≥ 3);- penetrating versus blunt trauma.A p-value less than 0.05 is considered statistically significant. If appropriate, predictive values between variables are calculated. Predictive values in continuous outcome variables are assessed using a multivariate regression model, and binary outcome measures are assessed using a multivariate logistic regression model. In case of binary outcome measures, predictive values are expressed as Odds Ratio's (OR) with 95% Confidence Intervals (CI). Data are analyzed using the Statistical Package for the Social Sciences (SPSS) version 18.0 SPSS Inc., Chicago, IL.Total-body CT scanning will be evaluated economically from a societal perspective against a conventional diagnostic strategy consisting of X-ray, FAST and selective CT scanning according to the ATLS guidelines. Cost-effectiveness analyses will be performed with the costs per patient alive and costs per patient alive without serious morbidity as outcome measures. Additionally, a cost-utility analysis will be done with the cost per QALY as outcome measure. Incremental cost-effectiveness ratios will be calculated, expressing the extra costs per (i) extra patients alive, (ii) extra patients alive and without serious morbidity, and (iii) additional QALY. Sampling variability will be accounted for by (bias-corrected and accelerated) non-parametric bootstrapping. Sensitivity analyses will be directed at applied QALY algorithms , unit costs of major cost components, and the (country-specific) friction period in case of production loss. Subgroup analyses will be performed by the predefined subgroups. The time horizon for the cost-effectiveness analysis will be six months following trauma. Because of this time horizon, no discounting will take place.The economic evaluation will take all direct and indirect medical and non-medical costs into account. The direct and indirect medical costs include the costs of initial trauma care, ICU-care and care at the general ward during the index admission - including all diagnostic and therapeutic procedures - as well as the costs of repeat hospital admissions, other intramural care like rehabilitation and extramural care during the first 6 months post trauma. Direct and indirect non-medical costs of, respectively, out-of-pocket expenses and production loss during the first 6 months will also be estimated. Volume data will be collected by case report form, institutional administrative databases and by patient questionnaires at 3 and 6 months, depending on the cost category. The patient questionnaire will be derived from the Dutch Health and Labour Questionnaire and adapted for international use. Unit costing will be based on activity based costing and hospital ledger data concerning the major diagnostic procedures in this trial. Unit costing of other health care components will be based on available national guidelines. In case of absence of national guidelines in specific countries, available unit costs from abroad will be recalculated using Organisation for Economic Co-operation and Development (OECD) purchasing power parities. Out-of-pocket expenses will be estimated as supplied by the patients. Indirect costs of production loss will be calculated according to the Dutch perspective by following the friction cost method, while applying the most recent friction cost period known at the time of analysis. Costs will be calculated for the base year 2012. Unit costs of other base years will be price-indexed.An independent Data and Safety Monitoring Board (DSMB), consisting of three members , is installed for this trial. On regular intervals, this committee will review accumulating trial data and provide advice on the conduct of the trial to the trial leader and Steering Committee. The DSMB will focus both on safety and effectiveness data. Standard Operating Procedures (SOP) will be used with respect to the schedule and format of DSMB meetings and with respect to the format and timing of presenting data. The DSMB can recommend the Steering Committee to terminate the trial when there is clear and substantial evidence of harm.The role of the DSMB is to perform an interim review of the trial's progress including updated figures on main outcomes and safety data. This review would include, but not be restricted to, the following:• monitor compliance with the protocol by participants and investigators;• monitor evidence for treatment differences in the main efficacy outcome measures;• monitor evidence for treatment harm ;• decide whether to recommend that the trial continues to recruit participants or whether recruitment should be terminated either for everyone or for some treatment groups and/or some participant subgroups;• suggest additional data analyses;• monitor compliance with previous DSMB recommendations;• consider the ethical implications of any recommendations made by the DSMB;• assess the impact and relevance of external evidence as supplied by the Chief Investigator.The DSMB will evaluate these safety and efficacy parameters at regular intervals. After 275 (25%), 550 (50%) and 700 (65%) included patients, non-blinded interim-analyses for evaluation of safety rules will be performed. No formal stopping rules based on statistical criteria alone will be used. The DSMB decides after evaluation of all necessary interim data whether the trial will be continued or terminated. Other investigators, designated by the Board of Direct of the AMC to control the trial will have the authority to gain insight in all the confidential data relevant for the trial as well.This trial is conducted in accordance with the principles of the Declaration of Helsinki , the MedTo participate in a research project the subjects must be volunteers and informed participants according to ethical principles stated in the Declaration of Helsinki. However, the acute life-threatening situation of severely injured trauma patients hinders a considered decision. Neither a legal guardian nor a legal representative of the patient can make a decision because of the time pressure or because they simply do not arrive in time. A temporary waiver of informed consent during randomization and the consecutive diagnostic phase during trauma survey was approved by the Medical Ethical Committee of the Academic Medical Center in Amsterdam. In all cases informed consent will be asked afterwards from the patient or the legal guardian/representative of the patient, as soon as reasonably possible.The need for prospective studies to measure the effect of immediate total-body CT scanning in trauma care has been stressed recently by several authors ,23,25,29The primary question that needs to be answered is whether immediate total-body CT scanning in severely injured trauma patients decreases mortality and significant morbidity when compared to conventional imaging strategies supplemented with CT. Therefore, randomization is within the hospital, ensuring that a comparison between imaging protocols is made per hospital instead of between hospitals. The design of the trial is multi-centered, with participating centers in The Netherlands, Switzerland and North America. This design assures that differences in trauma populations, trauma mechanisms and workflow in different parts of the world are taken into account as well. This is important to make sure that if an effect on outcome is seen that this can solely be attributed to the usage of a total-body CT scan.The in- and exclusion criteria assure that only potentially severely injured trauma patients are included and over triage is minimized. Especially severely injured patients are thought to benefit the most from fast and detailed information that becomes available with total-body CT scanning. Selecting the right patients for immediate total-body CT scanning is therefore crucial. Since the excluded trauma patients will be registered as well, final analysis will show whether the chosen inclusion criteria led to an appropriate selection of patients. Furthermore, severely injured patients are those patients in whom the radiation dose may be justifiable since their possible life-threatening injuries require accurate treatment as fast as possible. Trauma patients are exposed to a great amount of radiation and it is well known that CT scanning is a significant contributor to iatrogenic radiation exposure . The meaAfter conventional imaging in terms of X-rays and ultrasound has been finished the trauma leader has to decide whether or not selective CT should take place. The ATLS guidelines provide some decision rules but to some extent it is susceptible to individual judgment. Experience of the trauma leader and local infrastructures may influence these decisions. Furthermore, the randomization between total-body CT and conventional imaging supplemented with CT within each center holds the risk of a learning curve experienced by trauma leaders. If the trauma leader suspects detecting more injuries with a total-body CT scan than was expected on clinical grounds, performing selective CT scanning in the conventional arm could become more easily accessible and may lower the possible differences in outcome between the study groups. That is why the indication for selective CT scanning in the conventional arm are pre-defined, based on combined local protocols of the participating centers. The standardization of the conventional arm will lower the aforementioned risks.This trial aims to determine the optimal diagnostic strategy for severely injured trauma patients in the ED. If immediate total-body CT scanning is found to be the best imaging strategy in severely injured trauma patients it could replace conventional imaging supplemented with CT in this specific group. This will probably minimize the total diagnostic work-up time of the initial trauma evaluation. How this reflects in outcome needs to be analyzed in this trial. Furthermore, severely injured patients are already likely to receive selective CT scanning after conventional imaging according to ATLS guidelines or according to local trauma protocols. Segmented CT scanning in these patients, added to the conventional work-up, will result in a high total radiation dose because of overlapping radiation fields. It could therefore even be possible that an immediate total-body CT results in a lower the total effective radiation dose compared to the conventional work-up with selective CT scanning .The trial not only focuses on clinical outcome in terms of mortality and morbidity. Since radiation exposure and cost-effectiveness will be taken into account as well, the REACT-2 trial will provide a detailed overview of considerations that should be taken into account when discussing the efficacy of immediate total-body CT scanning in trauma patients. The large sample size will make sure that results are reliable and can be generalized to all international trauma populations and centers.http://ClinicalTrials.gov/NCT01523626 to compare immediate total-body CT scanning during the primary survey of severely injured trauma patients with conventional imaging strategies supplemented by selective CT scanning.The REACT-2 trial is an international multicenter randomized clinical trial The REACT-2 inclusion has started in April 2011. Results are expected in mid 2014.ATLS: Advanced Trauma Life Support; AIS: Abbreviated Injury Score; AMC: Academic Medical Center; ED: Emergency Department; FAST: Focused Assessment with Sonography for Trauma; GCS: Glascow Coma Scale; ICU: Intensive Care Unit; mGy: Milligray; ISS: Injury Severity Score; mSv: Millisievert; REACT-2: Randomized study of Early Assessment by CT scanning in Trauma patients -2; CT: Computed Tomography.J.C. Sierink, MD, is a Ph.D.-student at the Trauma Unit Department of Surgery, employed by the AMC Medical Research B.V., and supported by an unrestricted grant from ZonMw, the Netherlands organisation for health research and development (grant number: 1711020323). All authors declare that they have no competing interests.JCS drafted the manuscript, TPS and JCG co-authored the writing of the manuscript. All authors participated actively in the design of the trial and critically appraised the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-227X/12/4/prepub


This review describes mechanisms and circuitry underlying combination-sensitive response properties in the auditory brainstem and midbrain. Combination-sensitive neurons, performing a type of auditory spectro-temporal integration, respond to specific, properly timed combinations of spectral elements in vocal signals and other acoustic stimuli. While these neurons are known to occur in the auditory forebrain of many vertebrate species, the work described here establishes their origin in the auditory brainstem and midbrain. Focusing on the mustached bat, we review several major findings: (1) Combination-sensitive responses involve facilitatory interactions, inhibitory interactions, or both when activated by distinct spectral elements in complex sounds. (2) Combination-sensitive responses are created in distinct stages: inhibition arises mainly in lateral lemniscal nuclei of the auditory brainstem, while facilitation arises in the inferior colliculus (IC) of the midbrain. (3) Spectral integration underlying combination-sensitive responses requires a low-frequency input tuned well below a neuron's characteristic frequency (ChF). Low-ChF neurons in the auditory brainstem project to high-ChF regions in brainstem or IC to create combination sensitivity. (4) At their sites of origin, both facilitatory and inhibitory combination-sensitive interactions depend on glycinergic inputs and are eliminated by glycine receptor blockade. Surprisingly, facilitatory interactions in IC depend almost exclusively on glycinergic inputs and are largely independent of glutamatergic and GABAergic inputs. (5) The medial nucleus of the trapezoid body (MNTB), the lateral lemniscal nuclei, and the IC play critical roles in creating combination-sensitive responses. We propose that these mechanisms, based on work in the mustached bat, apply to a broad range of mammals and other vertebrates that depend on temporally sensitive integration of information across the audible spectrum. Our ability to perceive the location and identity of sound sources depends on information distributed across the frequency and time structure of complex acoustic signals. The peripheral auditory system performs an initial spectral analysis that separates acoustic information into a series of frequency channels. Subsequent analyses by the central auditory system combine information obtained from different frequency channels, including information about signal elements that have occurred at different times. The process of comparing information across frequency and time is termed here “spectro-temporal integration.” Spectro-temporal integration is essential for localization of sounds . This species displays two highly developed acoustic behaviors—echolocation and social communication—that require spectro-temporal integration for the analysis of its complex vocal signals. Spectro-temporal integration is particularly evident in the specialized responses to the mustached bat's echolocation signal  combination-sensitive response properties observed in midbrain and forebrain neurons depend on spectral convergence at distinct sites within the ascending auditory pathway, (2) spectral integration in combination sensitivity involves projections of low-frequency-tuned brainstem auditory neurons onto neurons tuned to much higher frequencies, and (3) glycinergic neurons are critically involved in both inhibitory and facilitatory combination-sensitive interactions. Further, the work identifies three regions that play key roles in creating combination sensitivity: the medial nucleus of the trapezoidal body, lateral lemniscal nuclei, and the inferior colliculus (IC). The integrative mechanisms described here are expected to apply broadly to vertebrates that utilize spectro-temporal integration to analyze complex vocal signals.This review first considers the auditory response properties of combination-sensitive neurons and then describes mechanisms and circuitry underlying these properties.Here we describe combination-sensitive response properties in the IC, the major nucleus of the auditory midbrain. The IC appears to be the locus of many of the integrative mechanisms underlying combination sensitivity. These mechanisms involve facilitation, inhibition, or both. Figure In the mustached bat's IC, the majority of neurons are combination-sensitive but estimates vary substantially across studies. Between 23 and 62% of tested IC neurons display facilitation, while 24–41% of tested neurons show inhibition without facilitation  component of bat sonar signals. For both low and high frequencies that evoke the strongest facilitation, we use the term “best facilitating frequency.” The best high facilitating frequency and the ChF were always very close . This indicates a special role of the fundamental in biosonar signal processing. The general view of this role is that the fundamental serves as an acoustic marker for the emitted sound: the fundamental in the emitted sound is sufficiently intense to activate auditory neurons but the fundamental in echoes is too faint to evoke a response  or CF3 (near 90 kHz) echo components. These neurons may be facilitated or inhibited by signals in the CF1 frequency range near 30 kHz or by signals in the FM1 range (29–23 kHz). These are designated CF–CF or FM–CF neurons, respectively. Analysis of FM echoes is performed by FM–FM neurons that are less sharply tuned to the frequencies in FM2, FM3, or FM4 sonar components. As described below, these differences in frequency tuning are correlated with differences in temporal properties. The neurons illustrated in Figure 1–FM3 type, responding to well-timed combinations of FM1 and FM3 elements of sonar signals  was unaffected by low-frequency duration  was unrelated to changes in signal duration, indicating that the facilitation has a fixed duration unrelated to the low-frequency signal duration  that display facilitatory combination sensitivity also show inhibitory combination-sensitive interactions  combinatorial interactions is in analyzing spectral content in social vocalizations. In several mustached bat social vocalizations, the energy in the frequency range below 30 kHz is variable  responses to ChF signals is inhibited by signals in the low (23–30 kHz) band; (2) this inhibition is usually phasic, locked to signal onset, and best when the ChF and low-frequency tones are presented simultaneously; (3) in many IC neurons, these inhibitory interactions co-occur with facilitatory interactions tuned to the same frequency bands. What mechanisms and circuitry underlie these features?Although inhibitory combination-sensitive interactions have been observed in thalamic  did not reduce the low-frequency inhibition, but additional blockade of glycine receptors (via strychnine) eliminated the low-frequency inhibition. Other tests  response. However, note that some features of the low-frequency inhibition, especially the inhibition that occurs at delays of 2–6 ms, are reduced by GlyR blockade. Across the sample of tested neurons, inhibitory receptor blockade reduced inhibition in many neurons but eliminated it in only a few neurons (12% of tested neurons). The reduction in inhibition can occur for several reasons: it may result from blockade of low-frequency-tuned inhibitory inputs, but it may also result from the overall increase in excitation to ChF tones that occur when most inhibitory inputs are blocked.Studies of IC neurons support a conclusion that inhibitory combination-sensitive responses arise at levels below the IC but may be modified by interactions within the IC  or direct glycine-evoked depolarizations as occurs in birds and developing mammals  of low-frequency input, Yavuzoglu et al.  placed aSeveral features of neurons in VNLLc are particularly well suited to the functional properties needed for facilitated neurons in IC. First, all of these neurons are thought to be glycinergic  inputs participate in response facilitation while the glutamatergic (“excitatory”) input conveys inhibitory combination-sensitive responses from INLL. This irony is a caution to circuit analyses: sources of “inhibition” and “excitation” to a particular neuron's response should be corroborated by mechanistic studies that establish whether inhibition or excitations are acting at that site.The interactions that create combination-sensitive responses in the mustached bat appear more hierarchical and the mechanisms appear more unitary than those observed in relation to some other complex response properties observed in IC, such as binaural processing or FM sweep selectivity  and that these projections occur in specific auditory brainstem nuclei. Thus, for combination-sensitive inhibition, low-frequency neurons in MNTB project to high-ChF neurons in INLL. For combination-sensitive facilitation, low-frequency-tuned neurons in VNLL project onto high ChF neurons in IC. These spectrally unmatched projections in the auditory brainstem have not been reported previously, but may underlie a variety of spectro-temporal integrative responses, from asymmetric inhibitory sidebands  and high-frequency neurons onto high-ChF neurons in IC. Projections from VNLL that are not matched to the ChF of target IC neurons may underlie the inhibition that contributes to FM sweep selectivity in other bat species . The mechanism underlying the timing of the facilitation is not understood, but our data suggest it does not depend on glutamatergic or GABAergic inputs to the IC facilitated neurons. We have speculated that the glycinergic rebound excitation may follow a variable period of increased chloride conductance that creates the opportunity for delayed low-frequency excitation. Further research is needed here, but it has the potential to reveal interesting postsynaptic mechanisms of information processing within IC neurons.The nature of facilitatory spectral integration by IC neurons in the mustached bat has led us to propose that individual neurons perform different signal processing operations in different cellular compartments, or processing domains Figure . SpecifiThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Aminoacyl tRNA synthetases (aaRSs) constitute an essential enzyme super-family, providing fidelity of the translation process of mRNA to proteins in living cells. They are common to all kingdoms and are of utmost importance to all organisms. It is thus of great interest to understand the evolutionary relationships among them and underline signature motifs defining their common domains.We utilized the Common Peptides (CPs) framework, based on extracted deterministic motifs from all aaRSs, to study family-specific properties. We identified novel aaRS–class related signatures that may supplement the current classification methods and provide a basis for identifying functional regions specific to each aaRS class. We exploited the space spanned by the CPs in order to identify similarities between aaRS families that are not observed using sequence alignment methods, identifying different inter-aaRS associations across different kingdom of life. We explored the evolutionary history of the aaRS families and evolutionary origins of the mitochondrial aaRSs. Lastly, we showed that prevalent CPs significantly overlap known catalytic and binding sites, suggesting that they have meaningful functional roles, as well as identifying a motif shared between aaRSs and a the Biotin-[acetyl-CoA carboxylase] synthetase (birA) enzyme overlapping binding sites in both families.The study presents the multitude of ways to exploit the CP framework in order to extract meaningful patterns from the aaRS super-family. Specific CPs, discovered in this study, may play important roles in the functionality of these enzymes. We explored the evolutionary patterns in each aaRS family and tracked remote evolutionary links between these families. Extensive studies have been carried out regarding their structures and functions et al.Here we studied the aaRS super-family using the Common Peptides (CPs) methodology. CPs are particular sets of deterministic motifs, composed of at least five consecutive amino-acids, selected by the Motive Extraction (MEX) algorithm (18). We exploited the CP framework for pursuing four objectives: (i) identify novel class-determining signature CPs, possibly bearing functional roles; (ii) elucidate remote homology among aaRSs and between aaRS and distant enzymes; (iii) uncover evolutionary patterns of aaRSs and (iv) identify CPs that reside on catalytic and binding sites. Class-specific CPs may be used to aid classification of novel aaRS sequences to these classes , Isoleucyl tRNA synthetase (IleRS), Leucyl tRNA synthetase (LeuRS) and Methionyl tRNA synthetase (MetRS). According to Woese It should be noted that out of the 11 CPs in CPs span a feature space in which the aaRS sequences are represented. Summing the occurrences of each CP over all aaRS family sequences, we obtained family-specific CP-based features. We next computed Pearson correlations between all pairs of aaRS families to obtain inter-aaRS similarity measures, correcting for CP overlaps across sequences . While tet al.α-helix (BFAH) structural features A striking difference in inter-aaRS similarity emerges when the correlations are calculated separately for each kingdom (mitochondria considered as a separate kingdom). Bacteria sequences comprise the majority of aaRSs sequences, resulting in similar inter-aaRS correlations . CorrelaCorrelations between TrpRS and TyrRS in Archaea are viewed also in Bacteria, but correlations between ValRS and IleRS are not apparent . These tLast, correlations between mitochondrial aaRSs exhibit a similar class I pattern as Bacteria with two exceptions: the emergence of correlation between TrpRS and IleRS, and the absence of correlation between TrpRS and TyrRS. In class II enzymes we observe correlations between PheRS and HisRS .Comparison with BLAST similarities reveals that many of the observed CP-space inter-aaRSs connections are not apparent using BLAST similarities with e-values below 0.01 or even as high as 1 . SpecifiIt has been demonstrated by In Ciccarelli's Tree of Life (ToL) et al. and Wolf et al.Since the majority of sequences are of bacterial origin, the majority of CPs are specific to the Bacteria lineage. Noted exceptions are the Seryl-tRNA synthetases (SerRS), with the majority of its CPs originating in the common ancestor of Archaea and Eukaryotes and CysRS, having comparable number of CPs originating in Bacteria and in the ancestor of Archaea and Eukarya. Both SerRS and CysRS exceptions are consistent with the observations of Woese Mitochondria are highly unusual organelles. They contain their own genetic material and protein-making machinery enwrapped in a double membrane. Mitochondria reside in eukaryotic cells, yet bear similarity to Bacteria in general and to α-Proteobacteria in particular et al.et al. postulated that the mitochondrial CysRS was retained, it follows from our analysis that even if this assumption is correct, the bacterial signal has eroded, suggesting a relatively higher mutation rate. Regarding the AsnRS and SerRS exceptions, indeed Brindefalk et al. reported that neither displayed characteristic bacterial or cytoplasmic patterns. However, while the CPs could not trace the α-Proteobacteria signal, we could still find remnants of the remote ancestry of Bacteria.We found good agreement between our findings and the phylogenetic analysis of Brindefalk Finally, eight aaRSs are reported to have a single eukaryotic aaRS nuclear gene in animals and fungi 15 CPs are found to be specific to Mitochondria . It is i−8, see −10). The known class I signature KMSKS and its modified motif KLSKR corresponding to the catalytic site were omitted from the analysis. The majority of the binding sites hit by the prevalent CPs are related to the esterification of the corresponding amino acids, including the intermediates of the aminoacylation reaction (e.g. Tryptophanyl-5′AMP – TrpAMP) or their non-hydrolyzable analogs (e.g. 5′-O-[N-(L-tyrosyl)sulphamoyl]adenosine - Tyr-AMS) and also adenosine triphosphate (ATP), while two CPs cover the binding domain to tRNA. Additionally, three CPs hit binding sites of Chloride and Magnesium ions (We looked for occurrences of our CPs in the protein sequences whose structures are deposited in Protein Data Bank (PDB) database ium ions . We thusBirA is a bi-functional protein, that acts as biotin-synthetase regulating its own transcription via binding to corresponding DNA regions. Structural similarity between its active sites and class II aaRS has been reported −5). We next retained four CPs prevalent in both aaRS and birA protein families, requiring appearance of each CP in a minimum of 20 sequences in both aaRSs and birA , leading to a list of 28 common CPs . It covers more than a hundred sequences from both aaRSs and birA, occurring mostly in ThrRS (131 out of 140 sequences in our dataset). Interestingly, the motif GIL[IVT]E resides on the binding site of biotin in birA (PDB IDs 1HXD We have employed the Common Peptides (CPs) methodology to carry out an analysis of aminoacyl tRNA synthetases (aaRSs), identifying novel class-determining signature CPs, elucidating remote homology between aaRSs and between aaRS and the birA enzyme family and uncovering evolutionary patterns of aaRSs and particularly in Mitochondria. We further showed that prevalent CPs are significantly associated with enzyme functional sites.We identified novel class I and class II signature motifs, that may aid future identification of these classes in newly found enzymes or Metagenomic data in an analogous way to Using the CPs as a feature space in which aaRSs are expressed, we were able to identify similarities between different aaRS amino acid sequences. Correlations, calculated on sequences belonging to species from each kingdom separately, reveal differences in correlation patterns unique to each kingdom. Comparison of the CP based similarities to ones calculated by BLAST sequence alignment show that CP-space presents a complementary view, providing inter-aaRS connections that are not reflected by conventional sequence alignment. In particular, some inter-aaRS similarities, e.g. class I LeuRS and TrpRS or similarity between class I ProRS and GluRS (the latter is based on a similarity in a specific domain), were unobservable in the BLAST similarity plot while other inter-aaRS similarities elucidated via the CP framework were observed only in few sequences from each aaRS family, making the identification a hard task.As shown in Last, we showed that prevalent CPs significantly overlap regions associated with catalytic activity and substrate binding in aaRSs with known 3D-structures, as well as identifying a motif that is common to the ThrRS and birA binding sites.In summary, CP analysis provides a complementary view on aaRS enzymes, their relationships with each other and their evolution, as well as possible relations with other proteins that are not included in aaRS families. Their overlap with functional sites on the enzymes emphasizes their biological importance and makes them candidates for further analysis that, focusing on them, may lead to novel insights on the mechanism and evolution of aaRS enzymes..We downloaded 5,406 protein sequences belonging to 22 enzyme families of aminoacyl tRNA synthetases (Enzyme Commission (EC) number 6.1.1.x) from the Enzyme and UniProt databases 1664 Biotin-[acetyl-CoA carboxylase] synthetases (birA) sequences were obtained from UniProt Binding sites were downloaded from PDB The datasets contain some highly similar enzyme sequences, belonging either to species of the same genus or different strains of the same species. In order to reduce redundancy we identified the groups of highly similar enzymes. This redundancy reduction was performed by first calculating BLAST pairwise sequence similarity et al.In order to create a unified CP-space in which all sequences were represented, we followed the procedure described by Gottlieb −5 to 1 produced similar results. Computing BLAST similarities between domains, we downloaded PFAM We used the Blastpgp program to find similarities between any aaRS sequence to all other aaRSs. Testing sequence similarities for E-values ranging from EThe Pearson correlation between different aaRSs were calculated based on the number of appearances of each CP on each aaRS sequences, normalized by the number of sequences of that aaRS. Some of the CPs overlap on some of the sequences . In order to decouple the appearances of two overlapping CPs, we created a merged CP whenever two or more CPs overlap on a certain sequence, resulting in additional 20,391 concatenated CPs. CP occurrences were thus counted for each CP only on sequences where it did not overlap any other CP, whereas concatenated CPs were counted when overlapping CPs were identified on a sequence. Correlations were thus calculated on the joint 31,003 features, including both 10,612 CPs and 20,391 merged CPs. A random model was constructed by shuffling 100 times the assignments of the CPs to different aaRSs, retaining the same number of appearances for each CP.The linking of species to the different kingdoms of life , was utilized by using the Kyoto Encyclopedia of Genes and Genomes (KEGG) Rickettsia, Brucella etc.), resulting in 38 different α-Proteobacteria species.Full names of α-Proteobacteria were downloaded from For each aaRS family, we extracted the list of CPs that hit only one of the three kingdoms of life  in that aaRS family, creating a list of CPs that are both aaRS- specific and kingdom-specific (but may occur in other kingdoms for other aaRSs). We next computed the hypergeometric enrichment of the CPs hitting the corresponding mitochondrial aaRS family in each of the kingdom-specific lists, correcting for false discovery rate (FDR) of 0.01.We identified 50 prevalent CPs, present in more than half of the sequences of at least one aaRS . From each aaRS family that is covered by these prevalent CPs, we selected the highest resolution PDB entry and looked for inclusion of the binding site in the CP, resulting in 29 exact hits. In order to identify additional eight CPs that reside in the vicinity of the biding site, we employed a looser threshold, requiring a maximal distance of three residues between the CP and the site.Figure S1Pearson cross-correlations of different aaRSs according to their shared CPs. Self correlations were left out for the purpose of clearer presentation.(TIF)Click here for additional data file.Figure S2Heat map of Pearson cross-correlations of different aaRSs according to their shared CPs in Bacteria. Self correlations were left out for the purpose of clearer presentation.(TIF)Click here for additional data file.Figure S3Heat map of Pearson cross-correlations of different aaRSs according to their shared CPs in Eukarya. Self correlations were left out for the purpose of clearer presentation.(TIF)Click here for additional data file.Figure S4Heat map of Pearson cross-correlations of different aaRSs according to their shared CPs in Archaea. Self correlations were left out for the purpose of clearer presentation.(TIF)Click here for additional data file.Figure S5Heat map of Pearson cross-correlations of different aaRSs according to their shared CPs in Mitochondria. Self correlations were left out for the purpose of clearer presentation.(TIF)Click here for additional data file.Figure S6BLAST Similarities between different aaRSs using PFAM domains. E-values> 0.01 are in blue.(TIF)Click here for additional data file.Figure S7BLAST Similarities between different aaRSs using PROSITE domains. E-values>0.01 are in blue.(TIF)Click here for additional data file.Figure S8Distribution of different aaRS according to the CPs appearing in all 3 kingdoms together (All), in Bacteria and Eukarya together (excluding Archaea) and in Eukarya and Archaea kingdoms exclusively.(TIF)Click here for additional data file.Table S1Occurrence of frequent CPs in aaRSs on sequences with a resolved structure.(PDF)Click here for additional data file.


Most preschool centers provide two 30-min sessions of gross-motor/outdoor playtime per preschool day. Within this time frame, children accumulate most of their activity within the first 10 min. This paper describes the design and baseline participant characteristics of the Short bouTs of Exercise for Preschoolers (STEP) study. The STEP study is a cluster randomized controlled study designed to examine the effects of short bouts of structured physical activity (SBS-PA) implemented within the classroom setting as part of designated gross-motor playtime on during-school physical activity (PA) in preschoolers.Ten preschool centers serving low-income families were randomized into SBS-PA versus unstructured PA (UPA). SBS-PA schools were asked to implement age-appropriate 10 min structured PA routines within the classroom setting, twice daily, followed by 20 min of usual unstructured playtime. UPA intervention consisted of 30 min of supervised unstructured free playtime twice daily. Interventions were implemented during the morning and afternoon designated gross-motor playtime for 30 min/session, five days/week for six months. Outcome measures were between group difference in during-preschool PA (accelerometers and direct observation) over six-months. Ten preschool centers, representing 34 classrooms and 315 children, enrolled in the study. The average age and BMI percentile for the participants was 4.1 ± 0.8 years and 69th percentile, respectively. Participants spent 74% and 6% of their preschool day engaged in sedentary and MVPA, respectively.Results from the STEP intervention could provide evidence that a PA policy that exposes preschoolers to shorter bouts of structured PA throughout the preschool day could potentially increase preschoolers’ PA levels.Clinicaltrials.gov, NCT01588392 Physical activity (PA) is critical to the healthy development of preschool-age 2.9-5 year) children. It is recommended that preschoolers should accumulate at least 120 min (structured and unstructured) of PA per day .9-5 year. Thus, aDuring a typical 30-min bout of gross motor playtime, direct observation and direct measures of PA (accelerometer data) indicate that preschoolers accumulate very few minutes of MVPA ,7. The aTo date, PA policies in preschool centers have focused on changing the outdoor playtime environment and little (if any) attention has been given to changing the delivery of PA in the academic classroom. In one of the few studies that examined classroom PA, Trost et al. found that the incorporation of a PA curriculum during classroom circle time significantly affected PA levels of children attending half-day preschool . Most loThe STEP study is a cluster randomized, controlled six-month trial to examine the effects of a SBS-PA program implemented during the designated gross-motor playtime during the preschool day in preschool-aged children. Outcomes of interest include total preschool-day PA, percent of time spent in MVPA, and sedentary time during gross-motor playtime and total preschool-day. Preschool centers were randomly assigned to one of two groups: SBS-PA or traditional unstructured PA (UPA). All children within a given preschool center were allowed to participate in the assigned intervention. This study protocol is in compliance with the Declaration of Helsinki and has been reviewed and approved by a University of Massachusetts, Amherst human research review board.A total of 15 preschool centers with a minimum of two full-day classrooms that serve low-SES areas in Springfield, Massachusetts area were approached to participate in STEP. Ten of the 15 preschool centers approached met the screening criteria and agreed to participate in the study. Reasons preschool centers declined participation included unwillingness to be randomized and inability to consistently provide 30 min of morning and afternoon gross-motor playtime per preschool day. Although not a requirement for eligibility, we assessed the existing PA policies for each of the 10 preschool centers enrolled in the study for randomization/stratification purposes. Existing PA policies were assessed using a modified version of the Environment and Policy Assessment and Observation Audit Tool (EPAO)[Within each preschool center, between 25 – 30 children were individually recruited for the assessment portion of the study. Children were eligible for the assessment portion of the study if they: 1) attended one of the 10 participating preschool centers; 2) were between 2.9 – 5 years of age on the date of baseline assessment; 3) did not have a condition limiting their participation in MVPA ; 4) unable to wear the activity monitor, and 5) attend preschool full-time. Children that were excluded due to eligibility criteria or whose parents did not wish to sign the informed consent were still allowed to participate in the intervention protocol; however no assessment data were collected on them.The STEP study utilized a cluster-randomized design because within a preschool center, classrooms sometimes share resources during gross motor playtime and thus, contamination between intervention arms would be unavoidable. Preschool centers were randomized into the treatment (SBS-PA) or control (UPA) group, stratified by preschool center size and existing PA policy. Randomization was performed using the Efron procedure .Due to the PA requirements set forth by the Massachusetts State Department of Early Education & Care, all participating preschool centers in the current study were mandated to provide 60 minutes of gross-motor playtime per day. Typically preschool centers divide this playtime into two 30-min sessions—one in the morning and one in afternoon. For this study, both the treatment and control interventions were implemented during the designated morning and afternoon gross motor playtimes five days per week for six months.The study intervention was developed based on the social ecological model and the Meta-Volition Model (MVM) . The MVMAdditionally, these theoretical models include personal factors: the children’s and teachers’ interest and participation in SBS-PA; the children’s belief that they have the ability to perform behaviors that will secure desired outcomes (self-efficacy); and outcome expectation (the intervention will result in more fun for the students and more manageable students for the teachers). Preschoolers have developed some aspect of self-efficacy because at this age they know that their personal action can produce certain results. Behavioral factors include the knowledge and skills available and needed to perform the intervention; and the degree of competence attained in using these skills. The structured activities utilized in this intervention will be developmentally appropriate for the motor skills of preschool-aged children. Of the three determinants of behavior development, environmental factors  will have the greatest relative influence on behavior in this intervention. With respect to this intervention, the social environment includes modeling by peers and adults (teachers). Preschoolers are able to pay attention and therefore peer and adult modeling of the intervention will enhance the children’s self-efficacy for learning and their ability to successfully perform the activities. The physical environment deals with the structurally integrated approach of the intervention . The intervention task has built-in reinforcement because the activities are designed to be fun.TFIR) was based on the Instant Recess® (IR) program, which has been utilized in different age populations [IR is 10-min physical activity routines, set to music with simple movements that engage major muscle groups. IR is available in audio (CD) or audiovisual (DVD) format, and is utilized to incorporate active breaks into routine organizational settings  for adults and youth. The SBS-PA intervention (TFIR) consisted of 10-min routines recorded onto DVDs that featured music and movements appropriate for preschool-aged children. A total of 16 audiovisual DVDs were created—ten existing IR routines that were originally geared toward adults (http://www.toniyancey.com) were modified for the current study, and six new TFIR routines were created by the research staff. The TFIR program was created in collaboration with a physical education specialist. All routines were designed to be simple, easy to learn and included low- to moderate-impact aerobic movements. Within each routine, movements (choreographed to music set at 100–120 beats/min) started with lower body actions before the addition of upper body movements. Each 10-min TFIR routine consisted of a warm-up (1 min), moderate- intensity activity (8 min) and a cool-down (1 min). Warm-up and cool-down movements were set to the same musical tracks, while the 8-min moderate-intensity movements were set to two 4-min tracks. Each TFIR DVD was accompanied with a movement-by-movement verbal and photo guide to assist classroom teachers in implementing the intervention.Tutti Fruitti Instant Recess . To implement the TFIR protocol, classroom teachers and students followed along with the TFIR DVD on a portable DVD player (which was provided to each classroom by the study staff for the duration of the study). TFIR DVDs were intended to be viewed by the classroom teacher while the students watched and followed the teacher’s lead. After the completion of the 10-min TFIR DVD, the students were allowed to engage in their usual gross motor playtime activities (unstructured play) for the remaining 20 min. The simplicity of the TFIR protocol enhanced teachers’ ability to implement the intervention. The TFIR protocol was implemented during the morning and afternoon designated gross motor playtime. Two TFIR DVDs (one for morning and one for afternoon playtime) were distributed to classroom teachers each week to ensure that preschool centers were implementing the same TFIR DVD during the intervention times. Each week, teachers were instructed to implement the assigned morning and afternoon DVD at the respective gross motor playtime. Each set of TFIR DVDs with its accompanying picture and verbal movement guide were dropped off the Friday preceding their implementation to give classroom teachers time to become familiar with the routine for following week. The previous week’s TFIR DVDs were picked up on Monday. Throughout the 6-month study, each morning and afternoon set of TFIR DVDs were repeated every eight weeks for a total viewing of three times.The The control intervention (UPA) consisted of traditional long bouts (30 min) of gross motor playtime and included supervised free time (unstructured) for the preschoolers to play on their own or with other children. During the gross motor playtime (either indoors or outdoors), children were provided with their usual play equipment, which was the current standard in the participating preschools. Children were allowed to choose from a variety of activities like running and jumping, bouncing, catching, and playing on structures or obstacle courses.TFIR protocol. During the training session, teachers were taught how to lead preschoolers in TFIR activities and were guided through three different TFIR routines. Training of the teachers from the control centers focused on the importance of allowing their students to play freely during the allocated gross motor playtime. All participating teachers were expected to follow their assigned protocols during designated gross motor playtime within each normal preschool day throughout the study. Research staff members were available throughout the 6-month protocol to assist classroom teachers in implementing their assigned intervention. At least once each week, research staff observed each participating classroom to determine if both the SBS-PA and UPA interventions were being implemented as designed.To enhance the fidelity to the intervention, within each preschool center all classroom teachers participated in a two hour in-service training session. The training sessions were held separately for staff at each preschool center. Training for teachers assigned to the SBS-PA intervention, included information on the importance of PA and how to implement the Participants total preschool day PA was assessed at baseline, and at 3-months and 6-months post-initiation of the intervention with the Actigraph accelerometer , which has been previously validated and used in preschool-age children ,24. The The Observational System for Recording Physical Activity in Children-Preschool Version (OSRAC-P) ,29 was uA self-report questionnaire, completed by parents/guardians, was used to obtain each child’s demographic and SES data. Standing height to the nearest millimeter (direct reading stadiometer) and body weight to the nearest 0.1 kg  were assessed with participants wearing light clothing, with shoes removed. Body mass index (BMI) was calculated as the weight in kilograms divided by the square of the height in meters. Parents/guardians reported the amount of time their child engaged in sedentary activity  using a self-reported survey ,30,31. Aa priori at a significance level of 0.25 or below) will be assessed univariately to determine which variables will be eligible for inclusion in the multivariable model. Intervention effects (change in PA) will be assessed with repeated measures mixed model analysis of variance or covariance, with childcare center as a random effect given that we do not expect that the within-group time trends will be homogeneous. Using a mixed effects model allows us to incorporate random effects for the center and class within center (clusters) and thereby model the correct covariance structure of the data and produce more accurate estimates of the effects under study and their standard errors. The distributions of individual variables will be evaluated and the data transformed as necessary to meet the assumptions of normality and homoskedasticity. For categorically scaled measures, we will use the generalized estimating equation methods for logistic regression analysis. Demographic variables will be added to multivariate models along with potential interactions with the intervention. Significant (at the p = 0.05 level) variables and interactions will be retained in the final model. For the baseline characteristics statistical comparison between groups was made with Wilcox Rank Sum tests for scaled variables and Chi-Square tests for categorical variables, with a two-sided alpha = 0.05. All analyses will be computed using SAS (version 9.2).The primary objective of the STEP study is to examine the effects of short bouts of structured PA to increase preschool-age children’s total school-day PA, as well as percent time spent in MVPA during the school day. The preschool center is the unit of randomization and intervention; the individual child is the unit of measurement. The analyses will use the child-level data, accounting for correlation of child responses within class and of classrooms within a center. For all analyses of primary outcomes and mediating variables we will use generalized linear modeling statistical methods. The methods allow assessment of a simple treatment comparison as well as multivariable modeling. Mediator and modifier significance . A total of 680 students from all preschool centers were eligible to participate in the study. Of those, 331 families responded to our advertisement and were eligible for participation. Prior to the initiation of the study, 16 participants were terminated from the preschools, for a total of 315 children enrolled at baseline. Baseline assessment was completed by 92% of eligible families. Baseline variables did not significantly differ between preschool centers for each group (SBS-PA and UPA); therefore their data were combined for this report. Baseline socio-demographic characteristics are presented in Table 2) did not differ significantly by group. The average BMI for boys and girls were at the 69th and 70th age- and gender-specific percentiles, respectively. Significant between group differences were observed for BMI percentile  The baseline prevalence of overweight (BMI ≥85th but <95th percentile) and obesity (BMI ≥95th percentile) in the SBS-PA and UPA groups were 19%, 12%, 22%, and 18%, respectively. No significant between group differences were observed for any PA variables. Participants in both groups spent 74.2% of their preschool day engaged in sedentary activity. Only, 6.4% of the preschool day (~9 h per day) was spent engaged in MVPA. During gross motor playtime, participants engaged in sedentary activity, light activity, and MVPA for 24.5%, 40%, and 33.6% of the observation intervals, respectively. There was a significant difference between the intervention and control groups for the observed intervals of light activity.Baseline characteristics of the sample are presented in Table In the past two decades, the obesity rate of preschool-aged children in developed countries like the United States has increased at an alarming rate ,35. BecaApproximately, 58% preschoolers in the US spend the majority of their day in an early childcare program  . TherefoIn order to change the PA policy in preschoolers to increase the amount of MVPA in which they engage, changes are needed not only in the types of PA to which preschoolers are exposed, but also in the academic classroom. The STEP study examines the effects of short bouts of structured PA implemented within the classroom setting as part of designated gross motor playtime on during preschool total PA and sedentary time in preschoolers. We hypothesized that short bouts of PA just prior to engaging in outdoor activity can not only increase classroom PA, but can also increase the amount of MVPA in which children engage during unstructured playtime.Of the 15 sites approached to participate in the program, we were able to successfully recruit 10 low-SES preschool centers. The established partnership with each preschool center allowed us to engage classroom teachers’ assistance in the recruitment of individual participants for the assessment portion of the study. The randomization process produced two groups with similar characteristics and only a few statistically significant differences. The number of significant differences identified between intervention and control groups in the current study could happen by chance. Most of the participants in the sample are of normal weight, however approximately 35% were classified as overweight/obese. The prevalence of overweight/obese status among preschool-aged children in the study sample is substantially higher than the reported prevalence (21.2%) in the 2007–2008 NHANES . We obseTFIR DVDs. These teachers who are usually pressed for time have reported an increased ability to use the TFIR DVDs to expose the children to more structured bouts of physical activity.On average the participants in this study spent a large percentage of the preschool day in sedentary activity and only 6% of the time engaged in MVPA. Their reported baseline MVPA (accelerometer) level is similar to what others have reported in preschool-age children ,29,45. OVery few studies have been successful at increasing PA during the school day in preschool-aged children. Several states are passing non-funded regulatory PA policy mandating that preschool centers should provide a minimum of 60 min of daily gross motor playtime for preschool-aged children. However, most of these policies do not indicate how this time should be allocated. Most preschools divide the 60-min block into two 30-min time blocks (one in the morning and one in the afternoon) of free playtime (unstructured). But this approach does not increase PA . ResearcBMI, Body mass index; EPAO, Environment and Policy Assessment and Observation Audit Tool; IR, Instant recess; PA, Physical activity; MVM, Meta-Volition Model; MVPA, Moderate-to-vigorous physical activity; SBS-PA, Short bouts of structured PA; SES, Socioeconomic status; STEP, Short bouTs of Exercise for Preschoolers; TFIR, Tutti Fruitti Instant Recess; UPA, Unstructured PA.The author(s) declare that they have no competing interests.SA conceived of the study and acquired funding. SA, ON, AM, and SS participated in data acquisition. SA, AL, and SS conducted the data analyses, however all authors participated in data interpretation. All authors participated in the design of the study. All authors were directly involved in drafting and edition of the manuscript. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/12/582/prepub


Rice tungro spherical virus (RTSV) and a DNA pararetrovirus Rice tungro bacilliform virus (RTBV). It is spread by insects owing to an RTSV-encoded transmission factor. RTBV has evolved a ribosome shunt mechanism to initiate translation of its pregenomic RNA having a long and highly structured leader. We found that a long leader of RTSV genomic RNA remarkably resembles the RTBV leader: both contain several short ORFs (sORFs) and potentially fold into a large stem-loop structure with the first sORF terminating in front of the stem basal helix. Using translation assays in rice protoplasts and wheat germ extracts, we show that, like in RTBV, both initiation and proper termination of the first sORF translation in front of the stem are required for shunt-mediated translation of a reporter ORF placed downstream of the RTSV leader. The base pairing that forms the basal helix is required for shunting, but its sequence can be varied. Shunt efficiency in RTSV is lower than in RTBV. But in addition to shunting the RTSV leader sequence allows relatively efficient linear ribosome migration, which also contributes to translation initiation downstream of the leader. We conclude that RTSV and RTBV have developed a similar, sORF-dependent shunt mechanism possibly to adapt to the host translation system and/or coordinate their life cycles. Given that sORF-dependent shunting also operates in a pararetrovirus Cauliflower mosaic virus and likely in other pararetroviruses that possess a conserved shunt configuration in their leaders it is tempting to propose that RTSV may have acquired shunt cis-elements from RTBV during their co-existence.Rice tungro disease is caused by synergistic interaction of an RNA picorna-like virus  Cauliflower mosaic virus, shunt-mediated translation downstream of the RTSV leader depends on initiation and proper termination of sORF1 translation and on formation of the basal helix of the downstream secondary structure. Given that RTBV-like shunt elements with identical sequence motifs are present in all RTSV isolates but absent in related picorna-like viruses, it is likely that RTSV could have acquired these elements after its encounter with RTBV. Alternatively, the RTSV shunt elements could have evolved independently to adapt to the rice translation machinery. Our study highlights on-going genetic exchange and co-adaptation to the host in emerging viral disease complexes.Ribosome shunting, first discovered in plant pararetroviruses, is a translation initiation mechanism that combines 5′ end-dependent scanning and internal initiation and allows a bypass of highly-structured leaders of certain viral and cellular mRNAs. Here we demonstrate that a similar shunt mechanism has been developed by the RNA picorna-like virus RTSV and the DNA pararetrovirus RTBV that form a disease complex in rice. Leader sequences of the RTSV genomic RNA and the RTBV pregenomic RNA possess a conserved shunt configuration with a 5′-proximal short ORF (sORF1) terminating in front of a large stem-loop structure. Like in RTBV and a related pararetrovirus  Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV). Individually these viruses exhibit rather mild symptoms: RTSV causes mild or indistinct symptoms, whereas RTBV infection causes yellowing and reddening of the leaves and results in stunted growth. The RTBV symptoms are accentuated in plants co-infected with RTBV and RTSV. Moreover, RTBV on its own cannot be transmitted from plant to plant, but it can do so with the help of RTSV that encodes an insect transmission factor cis-acting elements and sequence motifs to adapt to the host cell machinery and to synchronize their life cycles. Our study provides initial evidence for this hypothesis.Rice tungro disease is a significant constraint for rice cultivation in South and Southeast Asia. It is caused by a synergistic interaction of two viruses, Waikavirus in the family Secoviridae of picorna-like viruses Secoviridae use internal initiation of translation and the IRES elements identified in short leaders of two distinct viruses from the family Potyviridae do not resemble each other and those of animal picornaviruses RTSV belongs to genus Tungrovirus in the family Caulimoviridae of pararetroviruses Badnavirus of the Caulimoviridae) which have similar organization of ORFs I–III RTBV is the only member of genus Cauliflower mosaic virus (CaMV) from genus Caulimovirus of plant pararetroviruses Translation of RTBV pgRNA is initiated by ribosome shunting that overcomes the obstacles of a 700-nt leader sequence with multiple sORFs and a stable stem-loop structure The shunt configuration comprising an sORF terminating in front of the stable secondary structure has been identified in the pgRNA leader of most plant pararetroviruses Our computer-aided comparison of the 697-nt RTBV and the 514-nt RTSV leader sequences revealed remarkable similarities, suggesting that RTSV has co-evolved ribosome shunting :in vitroBoth leaders are unusually long, they contain several sORFs with a total number of 13 and 7 AUG codons, respectively, and can potentially fold into a stable stem-loop structure . Although the RTSV structure is more branched, its bottom section is rich in GC base-pairs which would ensure the stability: indeed this section is present in all optimal and suboptimal structures predicted by MFOLD, whereas the upper part of the structure can potentially assume several different conformations , A and BGAG UAG UCG). In CaMV and RTBV, the sORF1 stop codon is a take-off site for shunting ribosomes and proper termination of sORF1 translation in front of the stem base followed by peptide release is required for efficient shunting AUG GCUUCA  and AUG GCCCUU , respectively; the contexts deviate from a strong plant context because they lack A at position −3 relative to the first nucleotide of the start codon, but still have G at position +4) and it is positioned at a similar distance from the downstream secondary structure. Thirdly, the sORF1-encoded peptides are 6 and 5 amino acid long, respectively, and have identical termini: methionine and alanine at the N-terminus and glutamic acid at the C-terminus. Since the sORF1 amino acid composition generally does not affect shunt efficiency in vitro and in plantaBoth leaders have a very similar 5′-proximal sORF (sORF1). Firstly, sORF1 terminates at a short distance  upstream of the stem base and a nucleotide context of the stop codon is identical  is UA-rich, which would ensure a low index of secondary structure. Unstructured nature of the shunt landing site is likely required for efficient resumption of scanning by shunting ribosomes. Furthermore, like in RTBV and CaMV, a presumptive shunt landing site in the RTSV leader contains a non-AUG start codon (AUA), which is located at a similar distance from the stem and followed by an additional in-frame non-AUG (AUU). Interestingly, both non-AUGs are in frame with the downstream AUG start codon of the polyprotein ORF, though unlike the RTBV AUU, the RTSV AUA and AUU codons are in suboptimal contexts. By analogy with RTBV, it is likely that one or both of these codons are inefficiently recognized by shunting ribosomes to initiate translation of N-terminally extended polyprotein. This hypothesis is further supported by an UUUCU) is located just downstream of the stem basal helix. By analogy with animal picornaviruses which contain a pyrimidine tract in their IRES elements just upstream of the initiation codon UUUCU motif.In both leaders, an identical stretch of pyrimidines  placed downstream of the RTSV leader sequence or its mutant versions was monitored. We followed the same experimental settings and protocols as those used previously in a comparative study of molecular mechanisms of the RTBV and CaMV shunting In rice protoplasts, both RTSV and RTBV leaders drove relatively efficient translation of the reporter ORF, although the RTBV leader allowed a 1.6-fold higher initiation rate. Confirming our previous results, knock out (KO) mutations of the start (AUG to UAG) or stop (UAG to UAC) codon of RTBV sORF1 drastically reduced translation . The samTo further verify that sORF1-dependent translation downstream of the RTSV leader is initiated by ribosome shunting and evaluate a contribution of linear ribosome migration, we used a 40-nt Kozak-stem (KS) sequence which forms a perfect, compact stem-loop structure and blocks linear migration of scanning ribosomes Taken together, we conclude that almost half of the ribosomes entering at the 5′ end of the RTSV leader and initiating translation of sORF1 are able to shunt over the structure and re-initiate translation at the 3′-end of the leader. Notably, like in CaMV and RTBV, this mechanism depends on proper termination of sORF1 translation in front of the structured region. Extension of RTSV sORF1 by the stop codon KO mutation should lead to termination at the in-frame stop codon located 10 triplets downstream, i. e. within the ascending arm of the structure. This would melt the stem basal helix and bring the terminating ribosome away from the take-off and landing sites.To test if the stem basal helix structure is required for RTSV shunting, twelve point mutations were introduced either in its 5′-proximal or 5′-distal arms, which would disrupt secondary structure, and the compensatory mutations in both arms, which would restore stable secondary structure . The basInterestingly, in the absence of KS, the mutations in the 5′-proximal arm nearly abolished translation (5% of the wild type level), whereas the mutations in the 5′-distal arm reduced translation to 36% of the wild type level. The latter mutations in the presence of KS nearly abolished downstream translation (5% of the wild type level) . This sucis-elements present in the CaMV landing site but absent in the RTBV one We have established previously that, in wheat germ extracts supporting efficient ribosome shunting driven by the CaMV shunt elements Taken together, we demonstrate here that translation initiation of RTSV genomic RNA is controlled by its long leader and mediated largely by sORF1- and stem basal helix-dependent ribosome shunting. Further research is needed to characterize this mechanism in more detail. But given the striking similarity of all the shunt elements in RTSV and RTBV and especially the identity of certain sequence motifs in the shunt take-off and landing sites, it is very likely that both RTSV and RTBV use a similar shunt mechanism.Our comparison of five isolates of RTSV  showed that the leader sequence is remarkably conserved with only 35 polymorphic positions including 33 single nucleotide substitutions and 1-nt and 2-nt insertions/deletions (not shown). Only four substitutions occur in the shunt elements – one in the shunt landing sequence between the pyrimidine stretch and the non-AUG codon and three in the stem basal helix primary sequence (but not in the secondary structure) . Notablycis-elements identified in this study for viral infectivity. Previously, it has been shown that sORF-dependent ribosome shunting is essential for infectivity of CaMV We conclude that the shunt elements are well preserved in all RTSV isolates, indicating their biological importance for the virus. When an infectious clone of RTSV becomes available it will be important to test the role of sORF1 and other Maize chlorotic dwarf virus (MCDV), the second recognized member of genus WaikavirusSecoviridae, Parsnip yellow fleck virus (PYFV), the only recognized member of genus Sequivirus, is most closely related to RTSV and MCDV Among other viruses of the family It is thought that the ribosome shunt mechanism in plant pararetroviruses has evolved in order to protect the viral coat protein-binding, secondary structure element located within the leader GGGGCGCGTGGTGGCGGCTGCAGCCGCCACCACGCGCCCCCG (the self-complementary KS sequence is underlined) into the Cla I site of the RTSV plasmids “Wild type”, “KO start” and “KO stop”. The KS sequence shown above was also introduced in the middle of the RTSV leader sequence (in place of a guanosine at position 255) by a PCR ligation method, similar to that which we described previously The RTBV leader constructs “Wild type” and “KO start” have been described earlier The RTSV leader constructs “Stem Disrupt L”, “Stem Disrupt R” and “Stem Restore L+R” were obtained using 353 bp and 200 bp synthetic DNA fragments of the RTSV wild type construct that contain sequences from Cla I to EcoRV and from EcoRV to Xho I, respectively, each with 12 point mutations shown in in vitro translation experiments, the T7 promoter was introduced just upstream of the RTSV full-length leader and their variants with the sORF1 mutations by subclonning the Cla I-Sph I fragment from the RTSV plasmids “Wild type”, “KO start” and “KO stop” in place of the corresponding fragment of the T7 promoter-RTBV leader-CAT ORF plasmid described previously For the O. sativa were prepared and transfected with plasmid DNA by a polyethylene glycol method as described previously 6 protoplasts were transfected with 10 µg CAT-expressing plasmid and 2 µg β-glucuronidase (GUS)-expressing plasmid or 5 µg green fluorescent protein (GFP)-expressing plasmid. The GUS or GFP plasmid served as an internal control of transfection efficiency. Following incubation for 19–24 hrs at 27°C in the dark, protoplasts were harvested, protein extracts prepared and assayed for CAT and GUS (or GFP) accumulation, as described previously Protoplasts from suspension culture of in vitro experiments were performed as described in detail earlier 7mGpppG (in 6-fold molar excess over GTP) by incubation with T7 RNA polymerase (Biofinex). The integrity of the synthesized transcripts was evaluated on a 6% denaturing polyacrylamide gel. Equimolar amounts of capped transcripts (0.5 pmol) were translated for 1 hour at 27°C in a wheat germ extract. Accumulation of CAT protein in translation mixture was measured in duplicate by CAT ELISA (Roche) as recommended by the manufacturer. For each construct, in vitro translation was performed at least three times with freshly prepared capped RNA, yielding similar results.The Secondary structures at 25°C were predicted using the MFOLD program . The most optimal and suboptimal secondary structures of the 515 nt RTBV leader sequence are shown in in vitro using chemical and enzymatic methods, though alternative conformations were also revealed in that study MFOLD prediction of RNA secondary structure has proven to be reliable. For example, an MFOLD-predicted, large stem-loop structure of the 612-nt CaMV leader has been largely confirmed Figure S1The optimal and suboptimal structures of RTSV leader. The optimal (A) and suboptimal (B) structures of the RTSV leader predicted by the Wisconsin GCG MFOLD are shown. Positions of the sORFs' start and stop codons are indicated in red and green, respectively. The stem basal helix is encircled. (C) The optimal structure of the RTSV leader with (on the left) and without (on the right) the Kozak-stem (KS) sequence insertion. The KS is encircled.(PPTX)Click here for additional data file.Figure S2The optimal structure of MCDV leader. The structure predicted by the GCG MFOLD is shown with positions of the sORF start and stop codons indicated in red and green, respectively.(PPTX)Click here for additional data file.


This special issue collates 22 papers  reporting on various aspects of serum autoantibody research ranging from the identification of novel disease-related autoantigens up to the clinical significance of autoantibodies.The recent assurance that indirect immunofluorescence (IIF) is the gold standard for anti-nuclear antibody (ANA) testing from the Task Force of American College of Rheumatology (ACR)  has led pros and cons of digital automated indirect IIF platforms for autoantibody detection in systemic rheumatological conditions. J. Voigt et al. report original data on the evaluation of ANA on HEp-2 cells using the EUROPattern Suite automated processing and interpretation system. This is the first time that original data on the performance characteristics of this platform have been published in the form of a full-length paper. Concordant results between visual and automated evaluation by the EUROPattern reached 99.4%. This supports the notion that a precise and reproducible differentiation of positive and negative samples tested by HEp-2 cell lines is met by the developed systems. Discrepancies between manual and automated pattern recognition is largely limited to serum samples with mixed ANA patterns, but the developers of those platforms assure the reader that they will soon overcome the current limitations. J. Damoiseaux et al. evaluated the first automated anti-neutrophil cytoplasmic antibodies pattern recognition system developed using the AKLIDES platform. Discrimination of C-ANCA and P-ANCA is satisfactory but the sensitivity on ethanol-fixed neutrophils needs further improvement. D. Roggenbuck et al. have provided an up-to-date overview of the data so far provided using the AKLIDES system and critically discuss the A considerable proportion of apparently healthy individuals have IIF-detected ANA targeting the dense fine speckles 70 (DFS70) antigen. The clinical interpretation of positive anti-DFS70 antibody associated pattern (DFS) tests has emerged as one of the most important problems that routine laboratories are faced with, as it clearly influences the specificity and the positive likelihood of the ANA tests. In their review paper, M. Mahler and M. J. Fritzler discuss this topic and also describe a novel immunoabsorption method that can block anti-DFS70 antibody reactivity. An original paper by M. Miyara et al. analyzed the clinical value of anti-DFS70 antibodies in patients subjected to routine ANA testing by IIF and concluded that systemic autoimmune rheumatic disorders are less prevalent in patients with the DFS pattern compared to patients with homogenous or other ANA IIF patterns.R. Yoshimi et al. review the current data surrounding the clinical relevance and the pathogenic significance of anti-Ro/SSA antibodies in systemic lupus erythematosus (SLE), Sjögren's syndrome (SS), and other autoimmune disease. An original paper by A. Wacker-Gußmann et al. provided data suggesting that foetal magnetocardiography can complement foetal echocardiography as a noninvasive approach to detect early electrophysiological signs of atrioventricular delay in foetuses exposed to maternal anti-SSA/Ro and anti-SSB/La antibodies. Anti-C1q antibodies have been detected in women with SLE who experienced failed pregnancy. An interesting study from Greek investigators demonstrated that anti-C1q antibodies cannot differentiate failed from normal pregnancies. The same investigators have found elevated levels of IL-15 compared to those with missed abortions and healthy intrauterine pregnancies, underlying the diagnostic potential of this marker. Another original study has assessed the clinical relevance of circulating glucocorticoid-induced TNFR-related protein ligand (GITRL) levels in patients with SLE. Chinese investigators found that GITRL levels positively correlate with anti-dsDNA titres and these levels were significantly higher in SLE patients with renal involvement and vasculitis compared to patients lacking these clinical manifestations. Another original article from a Chinese group has found that the titres of carbonic anhydrase III and IV autoantibodies are unusually high not only in patients with SLE and rheumatoid arthritis, but also in patients with other diseases including type 1 and type 2 diabetes. P. C. Teixeira et al. review the literature and discuss the current knowledge on the role of autoantibodies against apolipoprotein A-1 in cardiovascular diseases. T. Shirai et al. provide an overview on the existing knowledge on the biological significance of anti-endothelial cell antibodies for vascular lesions in autoimmune rheumatic diseases. They also discuss in great detail the principles and applications of identifying autoantigens expressed on cell surfaces, known as serological identification system for autoantigens using a retroviral vector and flow cytometry (SARF). A. Shimatsu and N. Hattori review the literature and discuss the diagnostic, clinical, and pathophysiological features of macroprolactinemia caused by high molecular mass complexes of prolactin with immunoglobulin G (IgG) and in particular anti-prolactin antibodies. In their research article, R. Fu et al. and co-investigators used a proteomic approach based on the membrane of bone marrow cells to identify the antigenic targets of autoantibodies detected in a subgroup of patients with immune-mediated pancytopenia.A significant contribution comes from L. Mihályi et al. Those researchers provide a meticulous overview of their 40-year long clinical experience in the diagnosis and management of patients with autoimmune bullous dermatosis. In their research paper, A. Patsatsi et al. found that titers of anti-BP180 autoantibodies relate with disease activity in Greek patients with bullous pemphigoid, while a clinical study by Dalmády and colleagues provided data suggesting that autoantibodies targeting mutated citrullinated vimentin may assist the diagnosis of psoriatic arthritis. U. Lindberg et al. found that IgA ANCA specific for bactericidal/permeability-increasing protein (BPI-ANCA) identifies cystic fibrosis patients with adverse outcomesand discuss the pathogenic potential of these autoantibodies. Helicobacter pylori (Hp) antibodies, blood gastrin levels and are diagnostically useful in the classification of gastritis. Their predictive value is comparable to that of histologically assessed gastric biopsies. A British-German collaborative study found that the presence of Crohn's disease-specific pancreatic autoantibodies targeting the zymogen granule GP2 is largely limited to patients with ileal involvement. A. Kempinska-Podhorodecka et al. report on the influence of immunogenetics and their close interaction with humoral markers of liver autoimmunity. These researchers found that polymorphisms of genes involved in TNF-receptor signalling and in particular those of the TNF-receptor-associated factor 1 (TRAF1) do not confer susceptibility to primary biliary cirrhosis (PBC). However, the GG homozygotes have significantly higher titres of PBC-specific autoantibodies directed against gp210 autoantibodies compared to AA homozygotes, suggesting that this gene may immunoregulate the persistence of gp210-specific B-lymphocytes. Another intriguing original paper by C. Radzimski et al. reports the development of a recombinant cell-based IIF assay which allows efficient determination of autoimmune hepatitis-specific autoantibodies against soluble liver antigen. These autoantibodies are important for the confirmation of the diagnosis in patients with suspected autoimmune hepatitis and cannot be detected in routine laboratories by IIF.Finally, four original papers report on the relevance of autoantibodies in autoimmune gastrointestinal and liver diseases. A. Antico et al. assessed the predictive value of combined testing of four serological markers in the diagnosis of autoimmune gastritis. These markers include anti-parietal-cell antibodies (PCA), anti-intrinsic factor antibodies (IFA), anti-The broad range of important topics discussed by the authors participating in this issue further underlines the notion shared by the editors of this special issue that serum autoantibodies remain as one of the most valuable tools for the study of autoimmunity –6.Pietro InvernizziXavier BossuytDimitrios P. Bogdanos