Objective: To survey the evolution over the past decade of attitudes and practices of obstetricians in maternal–fetal medicine fellowship programs regarding the management of human immunodeficiency virus (HIV)-infectedpregnant women.Methods: Directors of all 65 approved maternal–fetal medicine training programs were sent questionnaires,responses to which were to reflect the consensus among members of their faculties. Programs were stratifiedbased upon the number of HIV-infected pregnant patients cared for in the previous year.Results: Responses reflect experience with over 1000 infected pregnantwomen per year, nearly one-quarter withadvanced disease. Combination antiretroviral therapy was prescribed by all respondents, universally in the 2nd and3rd trimesters. A three-drug regimen (often containing a protease inhibitor) was used more often by those whotreated at least 20 HIV-infected pregnant patients per year than by those programs seeing a lower number ofpatients (80 vs 59%).Despite the known and unknown risks of the use of antiretrovirals during pregnancy, only halfof all responding programs report adverse events to the Antiretroviral Pregnancy Registry; reporting was morecommon among the institutions seeing a higher number of patients (61 vs 45%). Seventy-eight percent of highervolume programs enroll their patients in clinical studies, usually multicenter, versus 35% of lower volumeprograms.Conclusions: Care for HIV² pregnant women has dramatically changed over the past decade. Antiretroviraltherapy is now universally prescribed by physicians involved in maternal–fetal medicine training programs.Given limited experience with these agents in the setting of pregnancy, it is essential for maternal–fetal medicinepractitioners to actively report on adverse events and participate in clinical trials.


Intrahepatic tumour is associated with alterations in splanchnic haemodynamics. To investigate the hypothesis that these are the result of a circulating vasoactive agent, rat small bowel segments were cross-perfused with arterial blood from groups (n = 12) of paired tumour-bearing (intrahepatic HSN sarcoma) and control rats. The vascular resistance of the segment was significantly greater during perfusion by tumour-bearing animals , suggesting that intrahepatic tumour may be associated with a circulating vasoactive agent. A similar mechanism may underlie changes in the hepatic perfusion index in patients with liver metastases.


Medicago truncatula roots, and quantified YFP fluorescence and mRNA levels. YFP fluorescence was normal in pOpOff2 transformed roots without dexamethasone but was reduced with dexamethasone treatment. Interestingly, dexamethasone removal did not reverse YFP inhibition. YFP expression in roots transformed with pER8 was low even in the absence of inducer. We used the dexamethasone system to silence acetyl-CoA carboxylase gene and observed prolific root growth when this construct was transformed into Medicago until dexamethasone was applied. Our study shows that dexamethasone inducibility can be useful to silence vital genes in transgenic roots.Understanding the functions encoded by plant genes can be facilitated by reducing transcript levels by hairpin RNA (hpRNA) mediated silencing. A bottleneck to this technology occurs when a gene encodes a phenotype that is necessary for cell viability and silencing the gene inhibits transformation. Here we compared the use of two chemically inducible plant promoter systems to drive hpRNA mediated gene silencing in transgenic, hairy roots. We cloned the gene encoding the Yellow Fluorescence Protein (YFP) into the dexamethasone inducible vector pOpOff2 and into the estradiol induced vector pER8. We then cloned a hpRNA targeting YFP under the regulation of the inducible promoters, transformed  Arabidopsis thaliana, could be transformed into an RPS2 plant by cloning the gene behind a chemically inducible promoterConditionally inducible vectors are useful for activating the expression of genes whose constitutive expression would have deleterious effects on the plant. For example the avrRPT2 gene, which in the presence of the RPS2 disease-resistance gene causes cell-death in The most common chemically inducible expression systems are comprised of two components; a chimeric transcription factor that is activated by the inducer and a promoter that is recognized by the chimeric transcription factor6810The estrogen inducible system employs a chimeric transcription factor XVE comprised of the DNA binding domain of the bacterial repressor lexA, the transcription activating domain of VP16 and the human estrogen receptor ERSteroid inducible promoters have been used to drive the expression of short interfering (si) RNA molecules produced by dicer like cleavage of double stranded RNAs (dsRNA). The siRNAs direct Argonaute nucleases to cleave endogenous transcripts with sequence homology to the dsRNAs, leading to the silencing of the endogenous transcripts. The pHELLSGATE transformation vectors were developed to allow hpRNA constructs to be generated using the Gateway recombination systemAgrobacterium rhizogenes transformation is an effective tool for investigating the functions of genes encoding phenotypes in plant rootsA. rhizogenes to introduce hpRNA constructs targeting candidate genes into roots16Agrobacterium rhizogenes mediated root transformation and monitored the kinetics and reversibility of YFP inhibition. We then showed that the dexamethasone system can be used to transform a hpRNA construction that targets a critical gene into Medicago roots and that chemical induction of this construct leads to a lethal phenotype. This study characterized the functionality of each modified system and tested their inducible silencing of a target gene, thereby providing a tool to investigate gene functions for root biology and fundamental RNAi process in plants.Petunia hybrida chalcone synthase A gene (CHS-A) intron behind the inducible promoter in each vector (M. truncatula roots by A. rhizogenes transformationWe modified the pOpOff2 and pER8 vectors to contain a Yellow Fluorescence Protein (YFP) gene transcriptionally regulated by the mas promoter to use as a visual markerh vector . These pTransgenic Medicago roots containing either pPopY, pPopY-hpYFP, pER8Y or pER8Y-hpYFP were treated with various concentrations of dexamethasone (Dex) or estradiol respectively, photographed YFP fluorescence daily, and fluorescence quantified using ImageJ. In the absence of Dex, the roots transformed with either pPopY or pPopY-hpYFP had similar YFP fluorescence levels , suggestWhen 1, 3 and 40 uM Dex was applied to pPopY-hpYFP roots, four days after induction fluorescence was reduced ~35%, 70% and 90% respectively relative to the absence of inducer , showingWe evaluated the changes in YFP mRNA levels by quantitative RT-PCR after treatment with the chemical inducers. Six days after the treatments with increasing concentrations of Dex, YFP mRNA levels were reduced up to 97% comparing with non-induced basal levels . To furtAs predicted from the fluorescence, there was a significant reduction in YFP mRNA in pER8Y-hpYFP roots even without chemical induction. However, the levels of YFP mRNA was further reduced in roots four days after the treatments with 1 and 10 uM estradiol .As indicated in To examine the reversibility of silencing after induction of the pPopY-hpYFP vector, we moved the roots to new media without inducer after 6 days’ treatment. YFP fluorescence did not recover up to 16 days after removal of Dex , althougTo evaluate YFP mRNA levels after Dex is removed, we isolated mRNA from induced roots at different time points after inducer removal and performed RT-PCR. For at least 16 days after removal of Dex, YFP mRNA levels remained at similar levels as before Dex removal, except for the slight elevation in message levels at day 16 . It is lWe are characterizing these vectors in roots with the goal of generating transgenic plants with RNAi constructs targeting genes that are critical for root survival. As a proof of concept, we inserted a RNA hairpin structure targeting cytosolic acetyl-CoA carboxylase (AccASE) into pPopY-hpYFP and transformed it into Medicago roots. AccASE catalyzes the ATP-dependent formation of malonyl-CoA from acetyl-CoA and bicarbonate and is a key enzyme for de novo fatty acid synthesisWe examined root growth rates in Medicago transformed with pPopY-hpACC or pPopY-hpYFP before and after Dex induction. Transformed Medicago roots were grown in non inducing media for seven days; then pPopY-hpACC transgenic roots and controls, i.e. pPopY-hpYFP, were moved onto media with or without 50 uM Dex for continuing growth and photographing roots daily. Notably, although most pPopY-hpACC transgenic roots are YFP positive (marked as pPopY-hpACC YFP+) and they did grow out few YFP negative roots (marked as pPopY-hpACC YFP−), indicating the growth of the sporadic non-transformed root cells. After moving to the new plates, the initial growth of all the genotype plants slowed in presence or absence of Dex. From 4–6 days, all the plants increased the growth rates. Markedly, along the monitored growth at different time points, Dex treated pPopY-hpACC YFP+ roots consistently revealed greatly reduced the growth rates compared to the treated pPopY-hpACC YFP− roots, the untreated pPopY-hpACC roots and to the treated pPopY-hpYFP controls , suggestTo validate the silencing of AccASE gene, we examined transcript levels of YFP and AccASE after the treatment of 50 uM Dex for 24 hours. Comparing with non-treated roots, Dex induced ~60% reduction in AccASE mRNA and ~85% reduction in YFP mRNA in pPopY-hpACC roots after 24 hours , suggestZuo and Chua (2000) summarized important traits for a chemical inducible systemIn the absence of Dex, Medicago roots transgenic for pPopY-hpYFP had similar levels of YFP fluorescence and mRNA as transgenic roots without the hairpin. In contrast, pER8Y-hpYFP transformed roots had reduced fluorescence and reduced basal mRNA levels compared to empty vector controls. Other researchers have reported on the leakiness of pER8 in the absence of inducer2223310Ideally chemical inducible promoters are sensitive to low concentrations of inducer and dosage dependent across a wide dynamic range. Previous studies showed GUS expression driven from pOp6 inducible promoter reached maximal levels 10 hrs after Dex treatment10In transgenic roots the pPopY-hpYFP vector was induced by Dex at concentrations ranging from 1 uM to 100 uM in a dose-dependent manner; no additional silencing was observed at higher concentrations . Our pPo10et al., 2005, i.e. unlike endogenous PDS gene, the silencing of LUC transgene was not reversed after removing Dex. Although the authors did not give reasonsA previous report of the Dex inducible pOp6 promoter showed reversal of the silenced PDS gene mRNA, but not silenced LUC transgene mRNA, within 10 days after Dex removalThe vector has a bidirectional pOp6 promoter that can drive transcriptions of two genes at once1010When roots were exposed to 3 uM or more Dex, YFP mRNA was depleted within 24 hours while no significant changes of YFP fluorescence was observed during the same period of time. These data suggested that the YFP protein half-life in Medicago roots is around 2.5 days. Using a similar approach, the half-life of GFP in C. elegans was estimated around 1.5 daysAlthough the application of dexamethasone system has been reported in leavesMedicago truncatula accession Jemalong A17 was obtained from Dr. Doug Cook . Agrobacterium rhizogenes strain MSU440 was used for root transformation.Seeds of the AflII site of pOpOff2 and the PvuII site of pER8. A hairpin cassette consisting of 373 bp of YFP sequences (base pairs 339–711) cloned as an inverted repeat flanking a CSHA intron was then cloned behind the inducible promoter in each plasmid. The GUS gene in the original pOpOff2 vector was deleted and replaced with a unique SfaA1 restriction enzyme site. The modified vectors were renamed pPopY-hpYFP and pER8Y-hpYFP respectively (The vectors pOpOff2ectively .Root transformations were performed as previously described using the A. rhizogenes strain MSU440http://rsb.info.nih.gov/ij).Transgenic roots were photographed under the dissecting fluorescent microscope with a Canon EOS 70D camera  and the intensity values (arbitrary units) of YFP fluorescence quantified by Image J software  adjacent to the YFP root ROI. Similar ROI calculations were done at different time points to quantitate YFP changes over time. The temporal changes in YFP fluorescent intensities were expressed relative to the basal YFP intensity prior to treatment.Transformed roots from pPopY-hpYFP or pER8Y-hpYFP system were induced by indicated concentrations of Dex or estradiol respectively and photographed for YFP fluorescence daily. Reversibility of induction was investigated by transferring pPopY-hpYFP roots onto media without Dex or with 50 uM Dex and photographed an additional 16 days.Medicago roots using the TRIzol reagent , treated with DNase1, and further purified using RNeasy Mini Spin columns . RNA (1ug) from each sample was converted to cDNA using High Capacity cDNA Reverse Transcription kit . 4 ng of reverse-transcription reactions was used for each RTPCR with primers (shown in Medicago Gene Index TC108192) and a ubiquitin carrier protein (Medicago Gene Index TC176441) as reference genes29−ΔΔCt method, was employed. Similar results were obtained for both reference genes  assay, total RNA was isolated from shown in  using ance genes , so the Root growth was monitored by marking the plate at the position of the root tips and then measuring growth every 24 hours using Image J (NIH). For each genotype, 20–40 independent root replicas were measured. The root growth rate was defined by root length growth divided by the time.t-test. Differences were considered significant at P < 0.05 and highly significant at P < 0.01.All the experiments were arranged in a complete randomized design with indicated independent replicates. Data are expressed as means +/− SEM for the roots from minimal three independent plants or as indicated. The data obtained were compared between either two different experimental conditions or two genotypes using a two-tailed Student’s How to cite this article: Liu, S. and Yoder, J. I. Chemical induction of hairpin RNAi molecules to silence vital genes in plant roots. Sci. Rep.6, 37711; doi: 10.1038/srep37711 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


According to clinical and pathological factors the prognosis of a patient with non-muscle invasive bladder tumors can be assessed. The prognosis is determined by the likelihood of recurrence(30-70%) and/or progression to muscle invasive bladder cancer(1-15%).Trans urethral resection of bladder tumors remains the initial therapy but adjuvant intravesical instillations are necessary.All patients benefit from a single immediate post operative instillation with a chemotherapeutic agent and for low risk tumors this is the optimal therapy.Patients with intermediate and high risk tumors need more intravesical chemo-or immunotherapy. Chemotherapy reduces recurrences but not progression. Intravesical immunotherapy(BCG) prevents or delays progression. Patients at high risk for progression may need upfront cystectomy.


A 61-year-old man with type II diabetes mellitus was referred with breathlessness onexertion. On auscultation, there was a continuous ejection systolic murmur on the leftupper sternal border. Transthoracic echocardiography showed a dilated vessel in aorticwall in the parasternal long axis view and a spPrimary coronary artery fistulas (CAF) are rare congenital communications between one ormore coronary arteries and a cardiac chamber or a great vessel. The RCA represents themost frequent site of origin of CAF in 60% of cases followed by the left coronary arteryin 35% while two CAF are uncommon (< 5%).The current case demonstrates the utility of CTCA in elucidating otherwise unusualtransthoracic echocardiographic appearances.


Polycyclic aromatic hydrocarbons (PAH) and their alkylated derivatives are generally mutagenic or carcinogenic compounds and are common trace components of environmental samples. Besides classical analytical methodologies such as capillary gas chromatography and liquid chromatography, high resolution spectrofluorometry (HRS) at low temperature in n-alkane matrices (Shpol’skii effect) is seeing an increasing interest –3.Since the first observation in the early fifties , this meT ≪ 77 K). The low temperature luminescence spectra exhibit a 0-0 transition with several sharp peaks (“quasi-lines”) having FWHM about 0.1 nm called multiplet structure and related to different substitution sites of the aromatic molecules in the n-alkane lattice [Fluorescence and phosphorescence spectra of aromatic compounds usually present broad bands at room temperature, having full widths at half-maximum (FWHM) of about 3 nanometers. A sharpening of the luminescence spectra is observed when PAH are incorporated into an appropriate n-alkane matrix frozen at low temperature  and the host  molecules seems to exist . When th−6 M and less), the formation of aggregates is minimized and the reproducibility of fluorescence intensity is not altered [A preliminary fast freezing of solution at 77 K in liquid nitrogen is also necessary to avoid aggregate formation . Another altered ,12.The American Environmental Protection Agency (EPA) has retained about 16 PAH as priority pollutants . They haMost of the EPA PAH could easily be identified and quantified by this technique. Low molecular weight aromatic compounds  were difficult to detect due to their low fluorescence quantum yield. To evaluate the accuracy of this method, we have analyzed several organic extracts  used for intercalibration exercises organized by the National Bureau of Standards . The results (imprecision less than 15%) obtained by Shpol’skii spectroscopy have been compared with values obtained by other analytical methodologies . As presented in Partial results presented here show the analytical capability of high resolution spectrofluorometry in Shpol’skii matrices for the determination of different priority pollutant aromatic compounds in the total organic extract of natural samples.


Myocardial salvage is currently being used as endpoint in several clinical trials and is determined by relating final infarct size to myocardium at risk (MaR). T2-weighted imaging (T2-STIR) cardiac magnetic resonance (CMR) has previously been shown to enable assessment of MaR up to one week after acute myocardial infarction. Recent experimental data indicate that the extent of MaR by T2-STIR varies over the first week which would have implications on how to design clinical cardioprotection trials using myocardial salvage as endpoint and in the clinical diagnosis of patients with myocardial infarction and normal coronary arteries.To investigate whether MaR as assessed by T2-STIR differs depending on scan day during the first week after the acute event in patients with reperfused first-time myocardial infarction.196 STEMI-patients from the MITOCARE and CHILL-MI trials undergoing acute percutaneous coronary intervention were included in the study. Eight additional patients with CMR on day 1 were also included. T2-STIR MR imaging was performed 1-7 days after the acute event and was used to evaluate MaR. Diagnostic quality on a scale from 0-3 and ability to correctly assign culprit vessel compared to angiography.There was no significant difference in MaR over the first week (p = 0.44, Figure Myocardium at risk by T2-STIR CMR imaging do not change in humans over the first week after acute reperfused myocardial infarction suggesting that MaR is a stable measure during this period of time.


Freeman-Sheldon syndrome (FSS) or “whistling face” syndrome is a rare congenital disorder complicated by characteristic facial deformities and muscular contractures. We report on a 64-year-old male patient presenting for surgical replacement of his aortic valve and review the available literature on anesthetic considerations and perioperative management principles. FSS frequently poses a significant challenge to airway management and gaining vascular access. Moreover, these patients are reportedly at risk for developing malignant hyperthermia (MH) or neuroleptic malignant syndrome.  Freeman-Sheldon syndrome (FSS) was first described in 1938  and is dWritten consent was provided for publication and photographs of this case.2) in combination with a severely decreased left ventricular function (left ventricular ejection fraction (LVEF); 25%). He had a known long-standing history of coronary artery disease and acute coronary syndromes and had undergone coronary stenting procedures repeatedly in recent years. Twice he suffered ST-segment elevation myocardial infarctions (STEMI). Admission chest X-rays showed cardiac congestion with bilateral pulmonary infiltrates. His blood work showed signs of systemic infection including elevated markers of inflammation and leukocytosis. After initiation of antibiotic and heart failure therapy his symptoms improved and he was scheduled for aortic valve replacement. A calculated EuroSCORE II of 2.08% lead to our center's interdisciplinary heart team decision for surgical aortic valve replacement (SAVR).Our patient was referred from another hospital with newly diagnosed critical aortic valve stenosis ; 0.7 cmHis past medical history was otherwise significant for type 2 diabetes, colonic diverticulosis, and COPD. During childhood and adolescence he had undergone numerous surgical procedures to correct skeletal deformities. Despite significant physical disabilities the patient had remained ambulatory and able to sufficiently perform activities of daily living (ADL) without assistance .μg) for additional analgesia and light sedation during intubation. Endotracheal intubation was successful without distress or hypoxemic events. After induction of general anesthesia (GA), direct laryngoscopy confirmed a Cormack Lehane 4 view. As alternate plan of securing the airway in case of failed AFOI or difficulties with ventilation, the attending surgeon remained in standby during induction to perform an awake tracheotomy in the spontaneously breathing patient.The patient did not report problems with previous anesthetics on preoperative evaluation. Written records of previous anesthetics could not be obtained. Given his orofacial anatomy including microstomia, retrognathia, and limited mobility of his neck, his airway was secured via awake nasal fiberoptic intubation (AFOI) . After lμg/h) infusions titrated to bispectral indices between 40 and 60. A trigger-free anesthetic was chosen to mitigate the risk for malignant hyperthermia (MH) or anesthesia-induced rhabdomyolysis.Prior to skin incision, cisatracurium (10 mg) was administered once. GA was maintained by propofol (4-5 mg/kg/h) and sufentanil  the patient was transferred to our intensive care unit sedated and intubated.Sedation was stopped on postoperative day (POD) 1. The patient initially presented disoriented with weak muscular tone and only minimal movement. During a spontaneous breathing trial at that time his respiratory mechanics appeared insufficient and labored and he was lacking sufficient cough and appropriate swallowing reflexes. Subsequently, light sedation with propofol was continued (Richmond Agitation Sedation Score- (RASS-) 1). Six hours later, the patient's sensorium and muscular tone had recovered enough to allow for safe extubation.During his postoperative course, creatine kinase levels were repeatedly measured and remained in low-normal range. Also, the patient never developed fever or acidemia.The patient was discharged from the ICU on POD 4 and left the hospital to rehabilitation on POD 7.To our knowledge this is the first report on the anesthetic management of an adult FSS patient undergoing cardiac surgery.While FSS remains a rare condition, the likelihood of perioperatively caring for adult patients with congenital pathologies will increase in the future. With an increasing life expectancy, ailments like cardiovascular disease become more prevalent and might require  interventions.Managing orphan diseases and congenital syndromes remain challenging. Most available literature to guide decision-making stems from pediatric patients. The fundamental topics for perioperative clinicians caring for FSS patients are the management of a difficult airway and vascular access . MoreoveEven though difficult airway anatomy is frequently encountered in patients undergoing thoracic surgery, patients with FSS almost invariably present with a challenging anatomy. Their small mouth opening and receding chin make oral intubation difficult and the limited nasopharyngeal space might render nasal placement of an adequately sized endotracheal tube impossible. A laryngeal mask airway can be a viable alternative in some patients without significant reflux disease or impaired gastrointestinal motility. AFOI is deemed best practice to secure these patients' airway. However, AFOI in FSS frequently can present a significant challenge, even for experienced practitioners .Establishing venous and arterial access can also be difficult in patients with contractures. The widespread utilization of ultrasound to visualize vessels has significantly improved success rates of vascular cannulation  Figures and 2.While regional and local anesthesia are considered ideal in these patients and recommended where possible  they areThe choice of sedative and anesthetic drugs is still debated notwithstanding that detrimental side effects seem to be rare. Benzodiazepines have been safely used as premedication although their intrinsic potential for muscle relaxation must be taken into account , 8. Ketaμ-receptor agonist, based on the visualized analogue pain scale.Several cases of hyperthermia possibly related to the use of anesthetics have been reported in FSS, and an inherent risk of MH cannot be safely confirmed or ruled out to date , 10. TheMetoclopramide has been linked to neuroleptic malignant hyperthermia in one patient with FSS, which was terminated by dantrolene . Given sAdult FSS patients undergoing cardiac surgery can be safely managed. Advance interdisciplinary planning and assignment of appropriate resources to the management of a potentially challenging airway and vascular access anatomy as well as planning for a prolonged ICU stay enable safe patient outcomes. A well-balanced, trigger-free anesthetic with short-acting opioids, limited use of muscle relaxants, and postoperative sedatives as well as neuroleptic drugs seem prudent in FSS.


The aim of the present study was to investigate various risk factors of suicidal behaviors, including mixed feature specifier, in Korean patients with bipolar disorder.We retrospectively reviewed medical charts from 2005 to 2014. A total of 334 patients diagnosed with bipolar disorder using DSM-IV TR were enrolled. The subjects were categorized into two groups according to history of suicidal behaviors and compared regarding demographic and clinical characteristics including mixed feature specifier. We re-evaluated the index episode using the DSM-5 criteria and classified into index episode with and without mixed feature. Logistic regression was performed to evaluate significant risk factors associated with suicidal behavior.Suicidal behavior had independent relationship with mixed feature at index episode using DSM-5 criteria and number of previous depressive episodes in Korean bipolar patients. The mixed feature specifier was the strongest risk factor in the present study.Suicidal behavior had independent relationship with mixed feature at index episode using DSM-5 criteria and number of previous depressive episodes in Korean bipolar patients. The mixed feature specifier was the strongest risk factor in the present study.


A 70-year-old male presented with direct trauma to his loin with gross hematuria, as an isolated case of blunt renal trauma (BRT) due to a traffic accident. A pre-existing renal lesion (PERL) was strongly suspected by his past history of gross macroscopic hematuria and monotrauma to the kidney without other associated injuries. Spiral CT scan with contrast and a retrograde pyelogram (RGP) confirmed an occult complex renal cyst. The gold standard of CT diagnosis in this situation is stressed. Computed tomography is particularly useful in evaluating traumatic injuries to kidneys with pre-existing abnormalities. The decision on the initial course of conservative management, ureteral retrograde stenting to drain extravasation, and its final outcome are discussed. Radical nephroureterectomy was carried out by a transperitoneal approach with an early vascular control of the renal pedicle. A brief review of recent literature has been undertaken.


CRISPR–Cas systems have provided revolutionary tools for genome editing. The discovery of Cas proteins with alternative activities has now enabled sensitive and robust tools for detecting nucleic acids. Recent reports harnessing these new CRISPR–Cas technologies display their potential for providing low-cost and practical diagnostic tools for pathogen and disease detection. Prokaryotic CRISPR–Cas  immune systems have offered a treasure trove of biotechnological tools over the past 5 years . Most ofThe origins of CRISPR–Cas systems as bacterial and archaeal defense mechanisms have led to an enormous diversity of Cas proteins owing to co-evolution between host cells and their invaders . The majcis with the target or in trans . TheTarget-activated nonspecific nucleases provide a robust mechanism for host defense, because Cas endonucleases can first sense invasive nucleic acid through specific target recognition and then amplify the signal through promiscuous collateral cleavage activity . SimilarIn early work aimed at developing a Cas13a-based RNA detection tool, East-Seletsky et al.  demonstrCas13a-based RNA detection opens the possibility of diagnostic applications through the sensing of nucleic acids associated with pathogens or diseases. However, useful diagnostic tools must be sensitive enough to detect very small amounts of nucleic acids, down to the attomolar range. To overcome the limited sensitivity of initial RNA detection tools, Gootenberg et al.  developeEGFR and BRAF genes.By coupling isothermal amplification to Cas13a target-activated reporter cleavage, SHERLOCK enabled detection of viral and bacterial nucleic acids with attomolar sensitivity . ImportaTrans Reporter), which couples isothermal amplification with Cas12a-based DNA reporter cleavage  molecules that are produced upon target-recognition by the class 1 Csm complex draws clear parallels to target-activated collateral cleavage by Cas12a and Cas13 , 10. GooThe early successes of CRISPR–Cas diagnostic tools present an exciting outlook for the future of this technology. The simplicity with which these tools can be reprogrammed makes them readily configurable for a huge variety of applications. Successful deployment of SHERLOCK in a paper-based lateral-flow format also demonstrates its capacity for easy application without the need for technical expertise or expensive equipment, similar to home pregnancy tests. However, it remains to be seen how effective these tools will be in clinical situations, especially in field conditions that could vary wildly from the laboratory settings in which the tools were developed. The use of RNA reporters for Cas13-based detection could present a potential limitation, because RNA is relatively unstable and susceptible to cleavage by cellular RNases. This could result in false positive results when using samples prepared in non-laboratory settings. It is possible that Cas12a-based diagnostics such as DETECTR [The discovery of new CRISPR–Cas systems has provided exciting opportunities for innovation over the past few years. Continued investigation of newly discovered systems will undoubtedly uncover more useful enzymes that may be implemented for improved sensitivity or stability of the current diagnostic tools. Nevertheless, the tools may already be mature enough for implementation and clinical testing. The potential to use these types of tests for rapid diagnostics could have an enormous impact in point-of-care settings, including early detection of viral outbreaks to ensure timely public health response. As with genome editing, these new CRISPR–Cas diagnostic tools are poised to revolutionize the accessibility of rapid, sensitive and accurate diagnosis of infectious and genetic diseases for people around the world.


Adult extracorporeal carbon dioxide removal (ECCO2R) systems and pediatric ECMO share the common objectives of having a low blood flow rate and low priming volume while safely maintaining sufficient respiratory support. The Hemolung is a highly simplified adult ECCO2R system intended for partial respiratory support in adult patients with acute hypercapnic respiratory failure. The objective of this work was to conduct pre-clinical feasibility studies to determine if a highly efficient, active-mixing, adult ECCO2R system can safely be translated to the pediatric population.14 healthy nonsedated juvenile sheep were used for acute  and 7-day chronic  in-vivo studies to evaluate treatment safety independently of respiratory related injuries. In all evaluations, we hypothesized that gas exchange capabilities of the Hemolung RAS in this model would be equivalent to the adult configuration performance at similar blood flows - minimum CO2 removal of 50 mL/min at a venous partial pressure of CO2 equal to 45 mmHg. Target blood flow rates were set to a minimum of 280 mL/min. Swan Ganz catheters were used under general anesthesia in the two acute subjects to evaluate blood gas status in the pulmonary artery.The Hemolung RAS was found to have adequate gas exchange and pumping capabilities for full respiratory support for subjects weighing 3 – 25 kg. The Hemolung device was estimated to provide a partial respiratory support for subjects weighing 27 – 34 kg. The seven-day studies in juvenile sheep demonstrated that veno-venous extracorporeal support could be provided safely at low flows with no significant adverse reactions related to device operation.The study outcomes suggest the potential use of the Hemolung RAS in a veno-venous pediatric configuration to safely provide respiratory support utilizing a significantly less complex system than traditional pediatric ECMO.


Cognitive impairments including deficits in working memory are commonly observed in schizophrenia. A bottom-up attentional bias has been suggested for encoding visually salient yet irrelevant information. To date it is not known if this bias persists when additional top-down information in the form of a predictive cue is provided. We were motivated to clarify this issue.40 patients with schizophrenia were measured and matched with 40 healthy control participants. During a change detection task four Gabor patches (two flickering and two non-flickering) with varying orientations were shown and participants had to memorize the orientations of the Gabor patches. A colored fixation cross was displayed before the stimuli either cueing two (predictive cue) or all four (non-predictive cue) Gabor patch locations resulting in a 2 x 2 design of four conditions with the factors salience (flickering vs. non-flickering) and cue (predictive cue vs. non-predictive cue). During retrieval a single Gabor patch was displayed, and participants reported if the orientation was the same or had changed in that location. At the beginning of each block participants were instructed to either encode the flickering or non-flickering patches (targets) whose location could either be cued or uncued. In 80 % of trials, a target was probed during retrieval.Patients encoded less information than healthy controls in all four conditions. Both healthy controls and patients encoded more visually salient information than non-salient information, and performance was near chance level during non-target trials. Patients encoded significantly more information when a predictive cue was provided before encoding visually non-salient information.Patients were able to overcome their bottom-up attentional bias of encoding visually salient irrelevant information when provided with top-down information. These findings are in line with previous reports of a bottom-up attentional bias during working memory encoding in schizophrenia. We propose that this bias can be overcome by providing additional top-down information.


A 45-year-old man presented with a thunderclap headache. Brain computed tomography (CT) scan without contrast showed massive subarachnoid hemorrhage (SAH). We transferred him to the angiography unit where he was diagnosed with a left (Lt) distal internal carotid artery (ICA) aneurysm. We secured the aneurysm using 8 coils. He was stable the following days. 1He was monitored via daily transcranial sonography (TCS) for the potential development of vasospasm. In TCS, the coiled distal ICA aneurysm was visualized as a round hyperechoic mass anterior to the midbrain. There was no aneurysm refilling on consequent evaluations. Unenhanced TCS is proposed as a screening tool for coiled aneurysm refilling.


Jdpd is an open Java simulation kernel for Molecular Fragment Dissipative Particle Dynamics with parallelizable force calculation, efficient caching options and fast property calculations. It is characterized by an interface and factory-pattern driven design for simple code changes and may help to avoid problems of polyglot programming. Detailed input/output communication, parallelization and process control as well as internal logging capabilities for debugging purposes are supported. The new kernel may be utilized in different simulation environments ranging from flexible scripting solutions up to fully integrated “all-in-one” simulation systems. Mesoscopic simulation aims at describing supramolecular phenomena at the nanometer (length) and microsecond (time) scale for large interacting physical ensembles (representing millions of atoms) within comparatively short computational time frames (hours) by “coarse grained” neglect of uninteresting degrees of freedom. Dissipative Particle Dynamics (DPD) is a mesoscopic simulation technique for isothermal complex fluids and soft matter systems that combines features from Molecular Dynamics (MD), Langevin Dynamics and Lattice-Gas Automata –5. It saWhereas DPD particles in general may be arbitrarily defined “fluid packets”  the MoleA simulation kernel software comprises the fundamental data structures and numerical calculation algorithms that are necessary to approximate the temporal evolution of a defined particle ensemble. DPD (as well as similar MD) code consists basically of a main loop over  successive simulation steps in which  particle pair force evaluations are the most time-consuming part , 14. ThuThe new Jdpd library enriches the small set of existing commercial , 16, opeDPD particle trajectories are guided by Newton’s equation of motion , 5:\docuDissipative  and random forces oppose each other and act as a thermostat conserving the total momentum and introducing Brownian motion into the system. The conservative forces comprise soft DPD particle repulsions (with a common cut-off length of 1 DPD unit), harmonic springs between bonded particles and electrostatic interactions between charged particles (the implemented model for the latter is an ad-hoc approach to take electrostatic long-range interactions between “a few” charged particles in the simulation box into account—details are outlined in  where anDissipative forceRandom forceapplied) . \documeharmonicBonds, dpdCutoff1 and electrostatics. An additional gravitational acceleration that acts on particle masses may be defined for every direction. All calculation classes for DPD and electrostatics forces and potentials extend abstract class ParticlePairInteractionCalculator of package interactions which itself implements a cut-off length based simulation box cell partitioning [getParallelisationSafeCellChunks of class CellBox in package utilities): These chunks guarantee a separated non-overlapping access of internal force array elements from parallelized calculation threads so that fast lock-free array manipulations become possible . An analogue lock-free parallelization feature with separated parallelizable bond chunks is realized within the bond related calculation classes (see package harmonicBonds).The sketched forces and corresponding potentials are implemented in calculation classes of the packages itioning : The resParticlePairDistanceParameters in package utilities). This caching avoids the time-consuming recalculation of particle pair distances (see relative performance factors of the different implemented integration schemata below): The saved number of expensively safe-guarded particle pair distance calculations may be  evaluated to be about 7 times the total number of particles in simulation for a common DPD particle density of 3. An upper bound for this empirical number may be deduced from the average number of all neighbor-cell particle pairs which is 40 times the number of particles in the simulation (which overestimates the number of relevant particle pair distances since—incorrectly—particle pairs with a distance above the cut-off length of 1 DPD unit are included):Another significant performance improvement is achieved by efficiently caching the already evaluated interacting particle pairs for reuse throughout different force calculations within a single simulation step (see use of caching class integrationType) that cover different integration techniques: (1) The original Groot-Warren scheme (GWMVV) [Since (unlike MD) dissipative DPD forces depend on relative particle velocities the common Velocity-Verlet (VV) integration of the equations of motion , 14 has  (GWMVV) , 28, 29  (GWMVV)  with an  (GWMVV) , 32 and  (GWMVV)  that reqDpdSimulationTask  that may be submitted to an appropriate thread executor service to be invoked. The DpdSimulationTask constructor requires six configuration objects: (1) An  RestartInfo instance that contains information about a possible simulation job restart , (2) an input instance that implements the IInput interface with all simulation settings , (3) an output instance implementing the IOutput interface for all output data, (4) a progress monitor instance implementing the IProgressMonitor interface for real-time simulation progress information, (5) an ILogger instance for log-level dependent accumulation of detailed internal calculation progress information and (6) a ParallelizationInfo instance that describes internal settings for parallelized calculations. All interfaces are located in package interfaces and possess concrete sample implementations like classes FileInput and FileOutput of package samples that implement file-based I/O methods. The RestartInfo and ParallelizationInfo classes are found in package parameters.A single simulation task is performed by instantiating a IInput object contains a Factory instance (located in package utilities) that implements enumerated type definitions for random number generation, DPD and electrostatics calculations, harmonic bonds or integration schemata. The factory pattern allows a simple extension or replacement of computational algorithms used throughout the whole simulation kernel, e.g. the implemented random numbers generators [Factory class. An IInput object also provides detailed molecular information (see class MoleculeDescription in package utilities): These molecular descriptions differentiate between topological bonds of a molecules’ particles for an adequate description of covalent connectivity and bonds between backbone particles that maintain a defined spatial 3D structure, e.g. ring systems or secondary and tertiary structures of proteins. As a matter of course both bond types are treated equally throughout calculations.An nerators –36 may bFileLogger and MemoryLogger in package logger) with different (extensible) log-levels to obtain detailed information about the simulation progress.For software programs with extensive parallelized numerical calculations the debugging facilities of current integrated development environments are often not sufficient to tackle subtle errors. Thus Jdpd comprises (extensible) loggers  but does not explicitly exploit specific hardware environments or devices like graphics processing units : AsJdpd is a new open DPD simulation kernel completely written in Java that complements the small available set of general purpose DPD kernels. It especially supports molecular fragment structures and offers parallelizable force calculation plus efficient caching options with an interface and factory-pattern driven design for comfortable and low expenditure code extensions, customizations or replacements. Detailed input/output communication, parallelization and process control as well as internal logging capabilities for debugging purposes are supported. The new kernel may be utilized in different simulation environments ranging from flexible scripting solutions up to fully integrated “all-in-one” simulation systems described in  where itThe Jdpd library uses the Apache Commons RNG libraries  and is p


After spinal cord injury (SCI), sensory feedback circuits critically contribute to leg motor execution. Compelled by the importance to engage these circuits during gait rehabilitation, assistive robotics and training protocols have primarily focused on guiding leg movements to reinforce sensory feedback. Despite the importance of trunk postural dynamics on gait and balance, trunk assistance has comparatively received little attention. Typically, trunk movements are either constrained within bodyweight support systems, or manually adjusted by therapists. Here, we show that real-time control of trunk posture re-established dynamic balance amongst bilateral proprioceptive feedback circuits, and thereby restored left-right symmetry, loading and stepping consistency in rats with severe SCI. We developed a robotic system that adjusts mediolateral trunk posture during locomotion. This system uncovered robust relationships between trunk orientation and the modulation of bilateral leg kinematics and muscle activity. Computer simulations suggested that these modulations emerged from corrections in the balance between flexor- and extensor-related proprioceptive feedback. We leveraged this knowledge to engineer control policies that regulate trunk orientation and postural sway in real-time. This dynamical postural interface immediately improved stepping quality in all rats regardless of broad differences in deficits. These results emphasize the importance of trunk regulation to optimize performance during rehabilitation. In healthy conditions, descending supraspinal commands continuously tune the dynamics of these circuits to ensure that movement-related afferent inputs adequately adjust gait patterns6. After spinal cord injury (SCI), the descending sources of modulation are severely disrupted. Consequently, sensory feedback signals become the primary source of control to produce and regulate leg movements after SCI10.Leg sensory feedback circuits play an important role in the generation and regulation of leg movements14. Repeated activation of sensory feedback circuits during standing and walking promotes activity-dependent reorganization of neural connections that ameliorates locomotor performance16. In particular, various studies showed that proprioceptive feedback circuits play a pivotal role in guiding motor execution and circuit reorganization after SCI. For example, epidural electrical stimulation of the lumbar spinal cord specifically modulates proprioceptive feedback circuits associated with extension and flexion of the legs, which enabled refined control of leg motor patterns during gait19. Moreover, we found that mice lacking muscle spindle feedback circuits fail to display the activity-dependent reorganization of neural pathways that support recovery after SCI20. These findings stress the importance of targeting proprioceptive feedback circuits in the design of rehabilitative strategies.The objective of gait rehabilitation therapies is to steer the functional reorganization of spared sensory pathways and residual descending projections through task-specific physical training in order to improve recovery24. In these scenarios, the trunk is typically constrained within bodyweight support systems providing vertically restricted forces, or exoskeletons that constrain pelvis movements. However, natural locomotion involves precisely-timed trunk movements in multiple directions25, which directly determine leg biomechanics, and consequently leg sensory feedback during locomotion29. Indeed, therapists commonly seek to adjust pelvis movements manually during rehabilitation. When possible, they also provide cues to the trunk in order to reinforce the interplay between trunk posture and leg biomechanics. However, the lack of technologies to assist these movements limits the spectrum of possibilities offered to therapists during rehabilitation. The development of rehabilitation protocols and robotic systems that actively regulate trunk posture during training relies on a deeper understanding of the interactions between trunk posture, proprioceptive feedback circuit modulations and leg motor pattern production during locomotion.The critical role of movement-related sensory information to steer recovery has motivated the design of training protocols, robotic interfaces and neuroprosthetic systems that predominantly focus on reinforcing reproducible leg movements during rehabilitationHere, we aimed to address these combined aspects in a well-controlled SCI rodent model of bipedal locomotion. First, we designed and fabricated a robotic postural interface that allows real-time control of mediolateral trunk orientation during locomotion in rats with severe spinal cord injury. This robotic system uncovered robust relationships between mediolateral trunk orientation and the bilateral modulation of leg motor patterns during locomotion. We next used a neurobiomechanical computational model of muscle spindle feedback circuits to study some of the mechanisms underlying these modulations. We found that mediolateral trunk orientation modulates the flow of information in muscle spindle feedback circuits. In turn, optimal locomotor performance emerged when mediolateral trunk orientation helped preserve the balance between muscle spindle feedback circuits associated with extensor and flexor muscles for both limbs. This knowledge guided the design of control algorithms that regulated mediolateral trunk orientation in real-time based on subject-specific deficits. Compared to static trunk orientation, this targeted strategy improved locomotor performance across a broad spectrum of gait asymmetries and motor deficits. These results provide an important proof of concept that stresses the need to develop similar dynamic trunk assistance during gait rehabilitation in humans.We developed a closed-loop robotic postural interface that supports the control of trunk orientation and postural sway in the mediolateral direction during bipedal locomotion in rats Fig. . To enab15  received a severe lesion of the spinal cord contusion that led to leg paralysis. To enable locomotion, we delivered an electrochemical neuromodulation therapy to the lumbar spinal cord according to methods described previously15 Fig. . Detaile15 Fig. .We then used this robotic postural interface to characterize the impact of trunk orientation on gait patterns. We mapped the relationships between trunk orientations  and the resulting changes in bilateral leg kinematics, muscle activity and ground reaction forces.Prior to stepping, we calibrated the attachment of each rat to the backplate ensuring that both feet touched the treadmill belt symmetrically. This posture was defined as the baseline trunk orientation.During stepping, rats exhibited variable gait asymmetries that emerged from well-known differences in the performance of the left and right legs after severe spinal cord injury. For example, Fig. Comparisons across rats (n = 5) with different lesion severities confirmed these results Fig. . RegardlWe next sought to study the neural mechanisms that were likely to underlie the corrections induced by trunk posture on gait patterns. Due to the extensive loss of supra-spinal control, sensory afferent feedback signals were the primary source of control and modulation of leg movements. In particular, muscle spindle afferent feedback circuits are known to play a critical role in the production of locomotion after SCI. These afferents directly encode the changes in limb abduction during trunk rotations. We thus investigated the impact of mediolateral trunk orientation on muscle spindle afferents firing during gait.18 to estimate the changes in afferent firing rates for multiple leg muscles. For each trunk orientation, we fed the recorded joint angle trajectories into a realistic 3D biomechanical model of the hindlimb30 and we estimated through inverse kinematic the corresponding muscle stretch and stretch velocity profiles for three pairs of antagonistic muscles acting at each joint of the leg   Fig. . We thenComputer simulations revealed that the trunk orientation profoundly altered the stretch profiles of all the simulated leg muscles, in particular those acting at proximal joints Fig. . At the The impact of trunk orientation on muscle spindle feedback circuits stressed the importance to optimally define trunk orientation, accounting for deficits that arise throughout movement execution. Commonly, optimal trunk orientations are set empirically by therapists at the beginning of each session, and then remain constant throughout training. We sought to regulate trunk posture automatically based on continuous monitoring of locomotor performance.We previously showed that trunk orientation monotonically modulates the abduction angles of the right and left legs, which in turn correlate with locomotor symmetry Fig. . We thusTo evaluate the performance of this control structure, we quantified its degree of controllability and stability Fig. . We set We then verified that the corrections of the controller converged towards optimal values for gait rehabilitation. We asked experienced trainers to fine-tune trunk posture for each rat during a training session in order to optimize locomotor performance, and compared the resulting trunk orientation with the rotation computed by the controller Fig. . DespiteWe finally aimed to capitalize on this robotic interface to restore postural sway during stepping. In healthy subjects, locomotion involves cyclic shifts in bodyweight, which are essential to reinforce loading during stance. The resulting afferent signals reinforce muscle recruitment and contribute to increasing lateral stability. These observations suggest that optimal gait patterns would emerge when reinforcing this aspect of gait at each phase of the locomotor movements.To test this hypothesis, we implemented an open-loop control logic that accommodated alternating changes in trunk orientation into the intrinsic stepping rhythm of each rat Fig. . SwayingCompared to static conditions, the delivery of dynamic swaying rotations in trunk orientation immediately increased the amplitude of vertical ground reaction forces and extensor muscle activity . Actuation was provided using a servomotor (Faulhaber 1028E012B) in combination with a zero-backlash gearhead (Faulhaber 12/5-161:1) which provided a theoretical output torque of 47.8 mN·m Fig. . This frThe control of the robotic postural system was implemented as a custom-made Simulink xPC Target application  that communicated with the sensing and actuation components via a Humusoft MF624 I/O board . To enable real-time control of posture based on the animal locomotor performance, we interfaced this application with a high-speed motion capture system  that monitored leg movement in real-time Fig. .Reflective markers (1 mm) were attached to bilateral hindlimb landmark joints, i.e. iliac crest, greater trochanter (hip joint), lateral condyle (knee joint), lateral malleolus (ankle) and distal end of the fifth metatarsophalangeal (MTP) joint. Raw 3D positions for each marker were continuously recorded (200 Hz) and imported into the Simulink application through an Ethernet connection (TCP/IP) using a dedicated datastream SDK library .Raw signals were filtered using adaptive filters (least mean squares), and processed online to account for missing data resulting from occlusions (interpolation from neighboring markers). Each marker was then assigned a specific label corresponding to the corresponding hindlimb joint. Custom-made rules ensured robust maker labeling along the limb. Transmission latencies through the complete loop remained within 20 ms.All further control policies were implemented as Simulink blocks within the xPC target application. A dedicated graphical user interface (GUI) allowed to modify control parameters, modes and targets during the experimental session.We sought to design feedback and feedforward control policies providing automatic adjustments to trunk postural orientation on the basis of online locomotor performance.To align postural changes to the stepping rhythm of each animal, the real-time system continuously monitored bilateral hindlimb kinematics and extracted key gait events (foot-strikes and foot-off events) Fig. . Foot-stFirst, we implemented a feedback  controller in order to correct asymmetric locomotor patterns and excessive abduction Fig. . At everSecond, we developed an open-loop feedforward control logic to enable rhythmic swaying rotations during locomotion Fig. . At pre-53 and validated experimentally54. We fed the computer model with crest, hip, knee, ankle, and metatarsophalangeal joint angle traces recorded experimentally in rats under different postural conditions (n > 10 steps) and we calculated the corresponding muscle stretch and stretch velocity profiles through inverse kinematics for 3 pairs of antagonist muscles, i.e., Tibialis Anterior (TA) and Medial Gastrocnemius (GM) for the ankle joint, Vastus Lateralis (VL) and Semi Tendinosus (ST) for the knee joint, and Gluteus Medius (GMed) and Ilipsoas (IL) for the Hip joint . Rats were housed individually on a 12-hour light-dark cycle, with access to food and water ad libitum. Animals were handled daily for at least two weeks before the surgeries. Animal care was performed twice daily throughout the post-injury period. All procedures and surgeries were approved by the Veterinarian Office of the canton of Vaud, Switzerland. All experiments and methods were performed in accordance with the relevant guidelines and regulations.17. Interventions were performed under aseptic conditions and general anesthesia (1–3% Isoflurane via facemask) on a heating pad to prevent hypothermia. Three animals received an incomplete lesion  and three additional animals received a complete spinal cord transection at the same level. One animal of this second group died right after surgery. A partial laminectomy was performed from T7 to T9 in which the spinous processes and the dorsal and lateral aspects of the vertebral column were removed to expose the spinal cord. The dura was opened along the mid-line. Contusions were performed using an Infinite Horizons Impactor (Precision Systems Instruments LCC) with special clamps attached to vertebrae T6 and T10. The spinal cord was impacted by a metal probe (1.3 mm diameter) with a force of 370 ± 30 kDyn. For complete transections, the exposed spinal cord was cut with customized tools. The extent of contusion lesions and the completeness of spinal cord transections were verified histologically post-mortem.All surgical procedures have been previously described in detailTo record electromyographic signals, bipolar electrodes were implanted into extensor  and flexor  muscles of the ankle joint. Recording electrodes were fabricated by removing a small part (0.5-mm notch) of insulation from the implanted stainless steel wire . A common ground was created by removing about 1 cm of Teflon from the distal extremity of an additional wire, inserted subcutaneously over the right shoulder. All electrode wires were connected to a percutaneous amphenol connector (Omnetics Connector Corporation) cemented to the skull of the rats. The proper location of the electrodes was verified postmortem.Stimulating electrodes were created by making a notch (~1 mm) in the insulation of Teflon-coated wires (same as for EMG recordings), which were subsequently secured during surgery at the midline overlying spinal levels L2 and S1 by suturing the wires to the dura. A common ground wire  was inserted subcutaneously over the right shoulder. All electrode wires were connected to a percutaneous amphenol connector (Omnetics Connector Corporation) cemented to the skull of the rat.Analgesia  and antibiotics  were provided for 5 days post-surgery.−1, ~40–60% BWS). Hindlimb locomotion was enabled by epidural electrical stimulation applied at S1 and L2 spinal segments  and systemic administration of pharmacological neuromodulators 7. Behavioral recordings started once the animals reached a stable locomotor performance (after ~21 days of training). Characterization and control experiments were performed on subsequent days.Starting 7–10 days post-injury, rats were trained for bipedal locomotion for 20–30 minutes every other day. Training took place on a bodyweight-supported treadmill , and Ranksum test or two-tailed Kruskal-Wallis test were used for nonparametric evaluations. The statistical a level P < 0.05 was considered significant.The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.Supp Video 1


Liquid biopsy technologies allow non-invasive tumor profiling for patients with solid tumor malignancies by sequencing circulating tumor DNA. These studies may be useful in risk-stratification, monitoring for relapse, and understanding tumor evolution. The quality of DNA obtained for these studies is improved when blood samples are collected in tubes that stabilizing white blood cells (WBC). However, ongoing germline research in pediatric oncology generally requires obtaining blood samples in EDTA tubes, which do not contain a WBC-stabilizing preservative. In this study, we explored whether blood samples collected in WBC-stabilizing tubes could be used for both liquid biopsy and germline studies simultaneously, minimizing blood collection volumes for pediatric patients.® tubes. Germline DNA was extracted from all blood samples and subjected to whole-exome sequencing and microarray profiling.Blood was simultaneously collected from three patients in both EDTA and Streck Cell-Free DNA BCTQuality control metrics of DNA quality, sequencing library preperation and whole-exome sequencing alignment were virtually identical regardless of the sample collection method. There was no discernable difference in patterns of variant calling for paired samples by either whole-exome sequencing or microarray analysis.Our study demonstrates that high-quality genomic studies may be performed from germline DNA obtained in Streck tubes. Therefore, these tubes may be used to simultaneously obtain samples for both liquid biopsy and germline studies in pediatric patients when the volume of blood available for research studies may be limited. Strategies for applying liquid biopsy assays to the treatment of pediatric solid tumors is still in the early stages of development –4. Studi® collection tubes from three patients.In pediatrics, the volume of blood drawn for research is sometimes restricted to ensure the safety of young children who have smaller blood volumes and may be more hemodynamically affected by sample collections. Thus, researchers must often choose between collecting samples for one research project over another. We anticipate that this may limit the usefulness of requesting that additional blood be collected in WBC-stabilizing tubes in ongoing research efforts in pediatric oncology. Therefore, we sought to determine whether samples obtained in WBC-stabilizing tubes could be used simultaneously for two purposes: 1) liquid biopsy studies and 2) germline studies. While the use of WBC-stabilizing tubes for ctDNA studies have been well-validated, to our knowledge there are no published studies describing the quality of germline DNA obtained from these tubes and their usefulness in performing germline genomic analyses. Here, we describe our experience performing whole-exome sequencing (WES) and genotyping by copy number and single nucleotide polymorphism (SNP) array analysis of germline samples obtained simultaneously in standard EDTA and Streck Cell-Free DNA BCT® tube.Blood samples were collected from patients enrolled on a Dana-Farber Cancer Institute IRB-approved banking study (DFCI protocol #11–104). Written informed consent was obtained from participants or parents of minor participants prior to study inclusion. Patients were selected for participation based on an expectation that they would have normal or near-normal WBC counts based on their clinical course. We also chose children older than 11 years old for the study to ensure that drawing multiple tubes of blood simultaneously would not infringe on the hospital established blood volume limits as it would in younger patients. We chose patients who were expected to require venous access for clinical purposes so that no additional procedures were required to obtain these samples. For each patient, 5 mL of blood was drawn into a purple top EDTA tube immediately followed by the collection of 10 mL of blood into a Streck Cell-Free DNA BCTBlood samples were processed at the Boston Children’s Hospital Biorepository on the same day as the specimens were acquired. For blood collected in both EDTA and Streck tubes, samples were first centrifuged at 1000 x g at 4° Celsius for 10 minutes in the collection tube. Plasma was then removed and stored for future use. The remaining blood, a combination of red blood cells and white blood cells, were then removed from the collection tube and subjected to DNA extraction using the Gentra Puregene Blood Kit (Qiagen) according to the manufacturer’s instructions. DNA was quantified for each sample using the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific).Control DNA for this experiment was obtained from the human B-lymphocyte cell line CEPH1408 (GM10831),  . A totalhttp://broadinstitute.github.io/picard/picard-metric-definitions.html). Reads were then aligned to the b37 edition of the human genome reference sequence (Human Genome Reference Consortium) using bwa (http://bio-bwa.sourceforge.net/bwa.shtml) utilizing the parameters “-q 5 -l 32 -k 2 -o 1”. Duplicate reads were identified and removed using Picard tools [https://www.broadinstitute.org/gatk/gatkdocs/org_broadinstitute_gatk_tools_walkers_indels_IndelRealigner.php). Base quality score recalibration was also performed using GATK  [Pooled sample reads were deconvoluted and sorted using Picard tools (rd tools . Alignmeon-bqsr) . Unalignhttps://trace.ncbi.nlm.nih.gov/Traces/sra/?study=SRP120033.Variant analysis for single nucleotide variants (SNV) was performed using MuTect v1.1.4 and annotated by Variant Effect Predictor (VEP). The SomaticIndelDetector tool from GATK was used for indel calling , 10. MuThttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE105032.Microarray analysis was performed on 1 ug of DNA sample. Each test sample was digested in parallel with a sex matched control  using the restriction enzymes Alu I and RSA I to obtain 200-500bp fragments. Following digestion, the test samples were labeled with Cyanine 5-dUTP and control samples were labeled with Cyanine 3-dUTP. The unincorporated dye was removed. Each test sample and correlating control were combined and hybridized to a custom 4x180K CGH plus SNP microarray . The copy-number probes on the array  have an average spacing of one probe every 35 kb throughout the genome and one probe every 10 kb in regions known to have clinical significance while the SNP probes  have an average spacing of 40 kb across the genome. Slides were scanned on a SureScan Microarray Scanner (Agilent) and data was analyzed through CytoGenomics v4.0.3.1.2 (Agilent) and Genoglyphix v3.1 (Perkin Elmer). Array data can be accessed at the NCBI Gene Expression Omnibus site Genomic DNA was obtained from blood collected in both EDTA and Streck blood collection tubes using a standard extraction kit with no modifications to the protocol for either tube. In all samples, we extracted over 100 μg of genomic DNA material using these standard procedures . In mostSequencing libraries were prepared from 100 ng of genomic DNA from each sample. All sequencing libraries were prepared and purified uniformly but with unique barcodes so that samples could be multiplexed for sequencing and then de-multiplexed during analysis. Library yields from all blood samples were remarkably similar and were comparable with the DNA library yields generated from DNA extracted from a human cell line, CEPH1408 . BioanalRecently published cancer predisposition studies have used both whole-genome sequencing and targeted sequencing approaches to identify recurrent germline variants in patients and families with a high incidence of cancer –15. TargThere were a similar number of total sequencing reads generated per sample . Plasma Sequence variants were identified by comparing each sample to DNA extracted from a human cell line, CEPH1408, which was sequenced simultaneously with the germline samples. A similar number of unfiltered variants were identified for each patient, regardless of whether the germline sample sequenced was collected in an EDTA or Streck tube . Since aDNA samples were also analyzed with genome-wide combined copy number/SNP mircroarrays. For each patient, regions of copy number alteration and homozygosity were identified and compared between samples obtained in EDTA tubes and Streck tubes. The quality control metrics were excellent for copy number analysis using the derivative log ratio spread (DLR) metric and SNP analysis using the call rate (CR) metric, regardless of how the sample was collected . Copy nuThe use of liquid biopsy assays that detect, quantify, and profile ctDNA are being routinely incorporated into clinical trials for adult cancers –24. Theshttps://clinicaltrials.gov/ct2/show/NCT02402244), the GAIN Consortium’s iCAT2 study , and the NCI-MATCH study for pediatrics .Such efforts in pediatric cancers are also beginning to emerge –4. PubliThere are several logistical hurdles to overcome in developing liquid biopsy technologies in trials enrolling pediatric patients at multiple institutions. One major issue is that samples must be collected and shipped to a central laboratory before being processed. ctDNA is easily extracted from plasma isolated from blood collected in EDTA tubes using standard extraction kits. However, several studies have demonstrated that plasma must be isolated from blood collected in EDTA tubes within eight hours of collection to prevent the chance of lysis of WBCs that will contaminate the plasma with genomic DNA –35. ThisOne competing research priority in pediatrics is the collection of a blood sample for germline DNA profiling. DNA for these studies are most often extracted from WBCs obtained from blood collected in an EDTA tube. A potential solution for collecting plasma for ctDNA studies while still prioritizing the collection of germline material for genetic studies would be to utilize a single tube of blood for both germline and liquid biopsy studies. To determine the feasibility of this approach, we collected blood from three patients simultaneously in EDTA and Streck tubes and performed WES and SNP-arrays on the matched germline DNA extracted from cells from each tube. We demonstrated that the WES and SNP-array data was of very similar quality for germline studies, suggesting that both ctDNA and germline study samples can be collected from a single WBC-stabilizing tube for pediatric cancer patients. Similar results were observed when utilizing column-based extraction methods to obtain genomic DNA from EDTA and Streck tubes (data not shown). One potential limitation of this study is that we were not able to test the effects of exposing blood samples to a wider range of temperatures or processing times. However, guidelines for handling Streck tubes suggest that samples be maintained at room temperature during shipment and processed within 7–10 days. We would expect that conforming to those guidelines would ensure the quality of cell-free and germline DNA extracted from blood collected in Streck tubes. We believe this data will facilitate the collection of liquid biopsy studies on multi-institutional trials without compromising germline studies or increasing the blood volumes requested for ongoing research.


Apis mellifera) collect and store both honey and pollen in preserved forms. Pollen storage involves the addition of honey or nectar and oral secretions to pollen granules. It is controversial whether the duration of pollen storage alters the palatability or nutritive value of the pollen storage medium. We examined how bees utilize different-aged stored pollen during an extended pollen flow. The deposition of pollen into wax cells and subsequent consumption were monitored daily on 18 brood frames from 6 colonies over an 8d observation period. Despite a greater abundance of older stored pollen cells on brood frames, bees showed a marked preference for the consumption of freshly-stored pollen. Two to four day-old pollen cell contents were significantly more likely to be consumed, while pollen cell contents more than seven days old were eaten at much lower rates. Similar experiments that controlled for cell abundance and spatial effects using cage assays yielded the same result. One day-old stored pollen was consumed approximately three times more often than 10d-old stored pollen, and two times more often than 5d-old stored pollen. These consumption preferences for freshly-stored pollen occurred despite a lack of clear developmental advantages. Young adult workers reared for 7 days on 1d-, 5d-, or 10d-old stored pollen showed no difference in body mass, stored pollen consumption, hindgut fecal material accumulation, or hypopharyngeal gland (HPG) protein titers, suggesting that different-aged pollen stores did not vary in their nutritional value to adult bees. These findings are inconsistent with the hypothesis promoting a period of microbially-mediated, “beebread maturation” that results in greater palatability or nutritive value for aged pollen stores. Rather, stored pollen that is not eaten in the first few days accumulates as excess stores preserved in a less preferred, but nutritionally-similar state.Honey bees ( Apis mellifera maintains large populations across periods of food scarcity by bulk preservation and storage of food materials [As a highly eusocial insect, the honey bee aterials ,2. Used aterials ,3–5. Duraterials –8. Antheaterials . Corbicuaterials .All collected pollen is packed into storage cells before consumption by colony members. Young adult workers consume over 80% of all stored pollen to complete their adult development and redistribute pollen to other bees in the form of nutrient rich secretions and semi-processed pollen fragments –16. YounLargely unknown is how honey bees consume stored pollen as pollen stores accumulate and age in the colony. Numerous authors have speculated that stored pollen undergoes significant microbially-driven nutrient changes during the initial phases of storage ,6,28–31.In this study, we examined how bees utilize different-aged stored pollens in the colony environment. We first tested honey bee consumption preferences for different-aged stored pollen produced over 8 days of storage. We focused on feeding preferences of young adult workers because these bees are overwhelmingly the primary consumers and redistributors of pollen resources for the colony –16,32,33A key prediction of the beebread maturation hypothesis is that older stored pollen is nutritionally superior to freshly-stored pollen. Because dietary pollen quality impacts adult development and function, we examined worker growth on pollen stored for different durations. We also examined the development of a tissue whose growth is known to depend on pollen availability. HPG produce proteinaceous glandular secretions used to feed developing larvae and other adult bees –14. HPG Apis mellifera linguistica) for all experiments and collections. All colonies were established in April 2014 from 1.4 kg worker bee packages headed by Italian queens . Each colony was maintained in a 10 frame single Langstroth deep box with a top feeder, entrance reducer, and bottom board . Colonies were kept in an apiary at the eastern edge of the Red Rock Agricultural Center  from colony establishment (April 2014) through use in field experiments (mid- to late-August 2014). The RRAC apiary was located in a Lower Sonoran desert creosote bush community consisting of rangeland bisected by drainage ditches and cattle pools [Larrea divaricata), triangle bursage (Ambrosia deltoidea), mesquite (Prosopis velutina), and acacia (Acacia spp.), with the latter two predominantly located along dry washes and pools.We used honey bee colonies headed by Italian queens  and distantly along the Santa Cruz River . However, no agricultural crops or weeds were in bloom at the main research station from colony establishment through the experimental period. Frequent observations of colony foragers revealed that workers foraged almost exclusively on native plants within 240m of the apiary during the month leading up to and including the experimental period. Workers engaged in water foraging gathered untreated surface water and/or ground water from ephemeral puddles, a water tank in the apiary, and a cattle pond located 380m from the apiary. Pesticide analysis of food stores from these colonies after the experiment and from colonies at this site in subsequent years revealed an absence of agrochemicals aside from Varroa mite treatment residues from previously-used comb. No chemical treatments were applied to the colonies from the time of package establishment through the end of the experiment. Representative samples of stored pollen and stored nectar/honey were removed from the colonies and analyzed for pesticide contents by the National Science Laboratory . 3 g of each material were pooled from 120–150 cells in 6 colonies. No pesticides aside from trace levels of the miticides Amitraz and Thymol were detected in these food materials in full pesticide panel screenings.Colonies were amply provided with food store frames and supplemental sugar and pollen before the experiment to support worker nutrition and limit foraging on distant agricultural fields. All colonies had excess stores of pollen and nectar present for at least three weeks prior to and through the experiment. Colonies were further supplemented with pollen patty (1:1:1 mixture (w/w) of dried corbicular pollen , MegaBee pollen supplement and sucrose ) and 2:1 sucrose:water sugar syrup from the start of the mid-summer dearth in early July 2014 up until 1 week before the experiment. By the beginning of the experimental period in mid-August 2014, each colony contained approximately 22,000 to 26,000 adult workers, 5–7 frames of brood, and at least 2200 cells of stored pollen. Colonies slightly increased brood production and net pollen stores through the 8d experimental period.Experiments were conducted in mid-August 2014 during the summer monsoon after four weeks of significant rains. Colony pollen collection was characterized by direct observation of foragers and monitoring of four sentinel colonies that were similar in size, food stores, and bee populations to the experimental colonies. Colony foragers collected polylectic mixtures of pollens that included at least 13 distinct plant species as determined by direct observation of foragers between the colonies and flowers . ColonieForagers were observed flying directly between the four sentinel colonies and these flowering plants. All colonies had foragers visit and manipulate the flowers of these plants. All plants were within 240m of the colonies. , 36Experiments 2 and 3 (described below) required pollen comb sections containing stored pollen of uniform age. These sections were obtained by restricting colony pollen deposition to a single frame for a limited time period (24h). Stored pollen was acquired from 6 colonies containing 20,000 to 32,000 workers and 4–7 frames of brood. Bees were housed in hive equipment consisting of (top to bottom) a top board, a top feeder, a Langstroth deep box , 10 fully drawn Langstroth deep frames (48.26 cm L x 23.18 cm H), an entrance reducer, and a bottom board .Pollen deposition was concentrated on comb sections by directing the access of pollen foragers to a single frame. First, an empty Langstroth deep box (“pollen deposition box”) was placed between the brood box and the bottom board. To keep returning foragers from entering the brood box, a rectangular wire mesh barrier with worker-excluding openings  was placed between the upper brood box and the lower pollen deposition box. A single Langstroth deep frame containing empty cell wax comb (“pollen deposition frame”) backed by Plasticell foundation  was placed in the pollen deposition box to provide empty cells for pollen deposition by the foragers. A second frame completely filled with nectar and capped honey stores was placed next to the pollen deposition frame to provide a sugar source for the foragers. Bee escapes were placed on the corners of the brood box to allow workers to leave to forage but prevent their return to the brood box . Returning foragers entered the colony through the entrance formed by the pollen deposition box, entrance reducer, and bottom board and deposited their corbicular pollen in cells on the pollen deposition frame. Pollen deposition occurred under these restricted conditions during a 24h period from 1000 hours to 1000 hours the following day. However, the bulk of the pollen deposition (over 96%) occurred in the early morning (0515 to 1000 hours) near the end of the 24h collection period.Once the pollen deposition period ended, the single-aged stored pollen frame was placed in a hive configuration that greatly limited further pollen deposition and oviposition. The pollen deposition box containing the single-aged stored pollen frame was moved to the top of the brood box. Further pollen deposition and oviposition on the single-aged stored pollen frame was restricted by the placement of a rectangular wire mesh barrier with queen excluding openings  between the upper pollen deposition box and the lower brood box. This barrier selectively allowed workers to tend the stored pollen but prevented the passage of queens or additional corbicular pollen into the pollen deposition box. Single-aged stored pollen frames were stored in this manner until use in bioassays.Single-aged stored pollen frames were cut into uniform-sized comb sections to provide food choices of standardized size and deposition age to bees in bioassays. Each frame was carefully cut with heavy shears into rectangular 8 cm H x 5 cm W sections before use in bioassays. Each comb section contained about 90 to 120 stored pollen cells out of the approximately 160 cells present. Honey and nectar cells on the comb were emptied by pipette and then cleaned of residues by selective exposure of cell contents to sugar-deprived workers.The deposition and emptying (consumption of cell contents) of stored pollen in storage cells was monitored by marking pollen cell changes on a transparency sheet placed over the frame face on a daily basis . During We readily recognize that this method of cell monitoring is highly conservative in that it only detects changes in pollen content at the whole cell level and underestimates small changes in individual stored pollen cells . Full st+) were at least 8d-old, but were otherwise indeterminately older. All other stored pollen cell contents were deposited during a known time interval and were referred to by their maximum possible “age” of pollen deposition from 1d-old to 7d-old.During the eighth and final monitoring time point (end of day 7 observation period), the total number of new stored pollen cells deposited (cells filled since the previous day’s observation) and emptied were tabulated by cell age for all monitored frames. Stored pollen cells marked on the first day with a black circle (8d+-old pollen cells) during this final interval.After the last marking time point, we calculated the number of stored pollen cells created and emptied (cell contents removed) during the last 24h interval. This set of markings was the only time point for which all stored pollen age classes were fully represented. The absolute numbers of stored pollen cells present, newly-formed (freshly deposited), and emptied (cell contents consumed) were tabulated for each of the eight age groups  would likely be consumed in greater numbers than less abundant pollen types based simply on their higher abundance on the frames. To obtain an accurate understanding of consumption preferences, we needed to correct for the relative abundance of each stored pollen age class. We calculated the relative pollen cell emptying rate (RPCER) for a given pollen age-class as:This metric readily detects over-emptying or under-emptying  of stored pollen cell age classes relative to the values expected if pollen cell emptying was random with regard to stored pollen age. No value could be calculated for emptying of 0d to 1d-old stored pollen since these stored pollen cells were emptied before their deposition was detected.One problem with our field assessments of worker stored pollen age preferences is that different-aged stored pollens vary in their abundance and spatial distribution on colony frames. Bees foraging for stored pollen on colony frames encountered many more older stored pollen cells than newer stored pollen cells based on numbers alone. To address these concerns, we conducted a series of dual choice assays where caged bees were presented with two different pollen comb sections of approximately uniform stored pollen cell age, size, and abundance.Dual choice assays were conducted with 3d- to 4d-old (post-emergence) adult workers because adult pollen consumption increases sharply just before this age . Each chad libitum on the two pollen sources for 20 hours. Each comb section was weighed before and after the assay. The total pollen consumed from each source was calculated as the difference between the pre- and post-assay masses of the source. Six cage replicates were performed for each of the three choice comparisons .In the dual choice assay, bees were given a choice between two different single-aged pollen comb sections (prepared as previously described). Each 8 cm H x 5 cm W comb section was placed vertically against the side wall such that the two sections were separated by the width of the cage. Bees were allowed to feed The development of young workers reared exclusively for 7 days on a single-aged stored pollen source was compared across three single-aged stored pollen treatments . Newly-emerged adult workers were collected from caged capped brood frames over an 8h period. Approximately 60 newly-emerged workers were placed in a Plexiglas cage as previously described. Each cage was provisioned with 20 mL 1:1 sugar syrup and 20 mL deionized water bottles. Four cage replicates were established for each of the three pollen treatments. All cages were maintained in an environmental chamber in the dark at 30°C at 35% relative humidity. Sugar syrup bottles were changed daily to avoid fermentation.The bees in each cage were provided with a pre-weighed pollen comb section containing either 1d-, 5d-, or 10d-old stored pollen (previously described). Each pollen source was provided in excess (90 to 120 pollen cells) to avoid overcrowding during feeding. The pollen source of each cage was replaced daily with new, preweighed material. The amount of pollen consumed was calculated as the difference between the pre-weight and post-weight masses of each pollen source.At the end of 7 days, the bees were anaesthetized with carbon dioxide and three bees were randomly selected from each cage for analysis of adult development. To obtain a more accurate measurement of worker body mass, we removed the highly variable gut contents of the honey stomach (crop), hindgut, and rectum before weighing the remainder of the body. The contents of these gut sections consist largely of food materials (sugar solutions (honey stomach) or feces (hindgut/rectum)) that otherwise inflate whole body measurements of worker mass. The whole body mass (wet weight) was measured immediately after removal of these contents. Hindgut and rectal fecal contents then were measured separately to provide an index of the amount of pollen consumed by the bee during adult development. Hindgut and rectal fecal contents have previously been used to estimate relative consumption of solid foods since adult workers do not defecate inside their nest (i.e. the cage) but accumulate feces in their hindgut and rectum . The fecEach bee head was then dissected and chemically analyzed to assess HPG development. Gland development was quantified as the total soluble protein content of macerated HPG tissue, a metric commonly used as a proxy for HPG size and quality ,37. SoluConsumption biases towards different-aged pollen stores were detected by comparing the observed consumption rate with the rate expected under random consumption. The overall distribution of emptied and unemptied cells across pollen-age classes was compared against the numbers expected by random emptying (consumption of cell contents) alone by a chi square test of independence. Post-ho+ older stored pollen cells. In the last observation interval, older stored pollen cells comprised 68.2% of the stored pollen cells emptied of their contents . Bees displayed significantly higher emptying rates of freshly-deposited stored pollen cells when cell emptying rates were adjusted for cell abundance on the frames. 2d-, 3d-, and 4d—old stored pollen cells were fully emptied at levels significantly higher than 7d-old and 8d-old stored pollen cells ). These newer stored pollen cells were fully emptied at rates 193% to 354% above predicted levels. By contrast, 7d-old and 8d+-old stored pollen cells were emptied at lower rates relative to their abundance. A critical change in cell content consumption patterns occurred between the 5d- and 6d-old stored pollen cells. Intermediate-aged 5d-old and 6d-old pollen cells were neither over-emptied nor under-emptied but appeared to represent a transitory phase in attractiveness to bees foraging for food within the colony.However, a different pattern of stored pollen consumption emerges among bees when the relative abundance of different-aged stored pollen cells is factored in. Bees emptied different-aged stored pollen cells at different rates  . Our locHow these feeding preferences contribute to mechanisms that regulate colony pollen use has yet to be explored. As individuals in a colony, honey bees must balance activities that result in pollen collection, pollen consumption, and brood rearing . Honey bWhile forager activities partially correct imbalances in pollen supply, worker feeding preferences shape the use of pollen once it is stored within the colony. However, the role of pollen consumer preferences in colony pollen utilization is less understood than foragers. Young nest workers that consume most stored pollen have different responses and olfactory sensitivities towards food materials than older workers, an attribute thought to contribute to age-related polyethism –75. The It remains to be demonstrated if the cues underlying these observed preferences are generalized cues or specific to this foraging system. Nonetheless, we have observed feeding preferences for fresh pollen in two locations on different types of forage at different times of the year . A key fS1 File(XLSX)Click here for additional data file.


This original clinical research study id focused on description of baseline anatomy and outcomes after transcatheter aortic valve implantation (TAVI) in patients presenting with severe aortic stenosis (AS) and bicuspid aortic valve (BAV). We compared this BAV population with a population of patients with AS and tricuspid aortic valves after a propensity score matching developed by a multivariate logistic regression according to a non-parsimonious approach. Baseline anatomical characteristics were obtained by transthoracic echocardiography (TTE) and multi-sliced computed tomography (MSCT) and compared by chi-square and t-student tests. Outcomes were evaluated by correct fisher test at in hospital and 30 days follow-up. We found that BAV patients presents more complicated baseline anatomy as compared to patients with tricuspid valves. These anatomical features lead to higher procedural complications as the need for a second device implantation. However this does not translate into increase in mortality rate at 30 days follow-up but rather correlate to a lower device success rate. Specifications TableValue of the data•Aortic stenosis in bicuspid aortic valve (BAV) remains a challenge for transcatheter aortic valve implantation (TAVI).•BAV patients presenting severe aortic stenosis are increasing in clinical practice•There is little evidence concerning TAVI in this population.•Our data can be an additional evidence for the feasibility of TAVI in BAV.•Indeed many questions are still open to optimize the sizing and find some dedicated devices for this population. Registries and sizing comparisons between operators could find a way to improve TAVI procedures in this subset.1–This study is a comparison analysis between patients with bicuspid or tricuspid aortic valves undergoing TAVI for severe AS.–Patients with AS and BAV present more complicated baseline anatomy as compared to patients with tricuspid valves.–These anatomical features correlate to a lower device success rate but are not related to increase in mortality rate at 30 days follow-up.–The lower device success rate reported was mainly related to a second device implantation need since nor PVL or mean gradient and mortality rate were significantly different.–Pre-procedural imaging and novel device technologies will help to address proper sizing and valve choice in the future.2From January to December 2016, 460 patients with tricuspid aortic valve underwent TAVI procedure in our institution for symptomatic severe AS.From January 2015 to April 2017, 83 consecutive patients with BAV had TAVI at our institution. The majority of these patients were indeed treated in 2016 (87%). Patients undergoing TAVI due to bio-prosthesis degeneration were not included.BAV were classified following the Sievers classification as reported in T test and categorical variables with a chi-square test. A propensity-score matching was applied to account for differences in baseline characteristics of both groups.Continue variables were compared using a Student’s A 1:2 propensity-score matching was performed on the basis of clinical risk factors for cardiovascular mortality and was developed by a multivariate logistic regression according to a non-parsimonious approach Baseline anatomical features and procedural characteristics were compared between the two groups as previously described. Outcomes were evaluated following the Valve Academic Research Consortium-2 definitions (VARC)-2 definitions. PPM values were classified into mild, moderate, severe and were analyzed at both in-hospital and 30 days follow-up The primary endpoint was all-cause mortality and early safety at 30 days. Secondary endpoint included device success.p Value ≤ 0.05. All results were obtained using the Statistical Package for the Social Sciences version 21.0 .Statistical significance was considered as


There are limited data on the cardiac effects of radiotherapy and chemo-radiotherapy on the heart in patients with non-small cell lung cancer (NSCLC). CART is a pilot and feasibility study designed to investigate change in myocardial function and identify changes in myocardial tissue properties in patients being treated with radical radiotherapy. Patients will undergo cardiac magnetic resonance (CMR) exam at baseline, during treatment, at 6 weeks and at 6 months after treatment completion. One component of a multi-parametric CMR protocol is contrast-enhanced first-pass perfusion assessment. Here we report our preliminary findings related to temporal changes in myocardial perfusion index (MPI).CMR was performed on a Siemens MAGNETOM Verio  3.0 Tesla scanner. First-pass myocardial perfusion was assessed by saturation recovery prepared dynamic contrast enhanced sequence during administration of 0.1 mmol/kg of gadoterate meglumine (Dotarem). Semiquantitative analysis was performed on segmented basal and mid-LV short axis slices to derive normalized upslopes of myocardial signal intensity profiles . The change in MPI over time was analysed statistically using a linear mixed effects model.13 patients currently have undergone CMR at baseline and during treatment , 8 patients have undergone CMR at 6 weeks post treatment initiation (5 patients lost to follow-up/ died), and 6 patients have undergone CMR at 6 months from baseline (n= 2 lost to follow-up/ died).Perfusion index Figure  varies sBased on our results, perfusion CMR is a feasible tool to assess changes in microvascular function in patients with NSCLC following radiotherapy/chemo-radiotherapy. We have found that the change in microvascular perfusion is most pronounced 6 weeks following the completion of treatment. This finding will be used in the design of future clinical studies, where measurement of MPI will provide a robust comparison of different existing and emerging treatment protocols for NSCLC with regard to their effects on myocardial physiology.