We present the case of an 88-year-old female who presented to the emergency department (ED) with suspected massive pulmonary embolism (PE) causing respiratory failure, right heart strain, and shock, who despite early and aggressive resuscitation with vasopressors and continuous peripheral infusion of tissue plasminogen activator (tPA), suffered a cardiac arrest in the ED. We describe the approach of a tPA bolus directed through a central venous catheter, resulting in return of spontaneous circulation and immediate improvement in physiologic parameters prior to confirmation of PE with computed tomography angiogram. We further hypothesize that in patients deemed too unstable to be transferred for embolectomy or catheter-directed thrombolysis, central venous catheter-directed bolus tPA may be more effective than peripheral infusion alone. Pulmonary embolism (PE) is a common life-threatening cardiovascular condition encountered by emergency physicians, with presentations ranging from incidental asymptomatic sub-segmental pulmonary embolus to massive saddle embolus and circulatory collapse. In general, thrombolysis and surgical thrombectomy are reserved for cases of massive PE, as defined by acute PE associated with persistent hypotension or shock. Thrombolytic regimens, however, vary among different studies with no clear recommendation on ideal dosage or route of administration. The American College of Emergency Physicians (ACEP) clinical policy on management of PE gives a level B recommendation to “administer thrombolytic therapy in hemodynamically unstable patients with confirmed PE for whom the benefits of treatment outweigh the risks of life-threatening bleeding complications,” and a level C recommendation to “consider thrombolytic therapy in hemodynamically unstable patients with a high clinical suspicion for PE for whom the diagnosis of PE cannot be confirmed in a timely manner.”2) 14, and oxygen saturation of 88% on 100% fraction of inspired oxygen (FiO2). Her pupils were equal and reactive, and her heart, lung, and abdominal exam was unremarkable. She had cool extremities with peripheral cyanosis but palpable distal pulses. No spontaneous extremity movements were noted. A 12-lead electrocardiogram (ECG) showed a right bundle branch block without signs of acute myocardial infarction and no prior ECG for comparison  following a respiratory arrest. The patient was at home and had a syncopal episode while getting dressed. Paramedics arrived to find the patient hypoxic and in respiratory failure. She was intubated after receiving 10 milligrams of diazepam intravenously and transported to our ED. Her pre-hospital capillary glucose measurement was 230 milligrams per deciliter (mg/dL). Her presenting exam was notable for an obese elderly female, intubated and sedated with heart rate of 92 beats per minute (bpm), blood pressure 53/40 millimeters of mercury (mmHg), respiratory rate 21 breaths per minute by manual bag, end-tidal carbon dioxide  piggy-back while a CVC was being prepared. A bedside echocardiogram showed significant dilation of the right ventricle .Given the sudden cardiac arrest with hypoxia, persistent hypotension, and the echocardiogram findings with no acute myocardial infarction on the ECG, the most likely diagnosis was thought to be massive PE. The patient did not have any significant response to the initial infusion of thrombolytics. A right internal jugular CVC was inserted under sterile conditions. Moments later, the patient became bradycardic and then pulseless. Cardiopulmonary resuscitation (CPR) was initiated and two minutes of chest compressions were performed to complete the first round. Subsequently, the decision was made to push a 50mg tPA bolus through the CVC. CPR continued for four additional minutes and the patient had a total of 1 milligram of epinephrine and 75 milliequivalents of bicarbonate given, after which return of spontaneous circulation (ROSC) was achieved. Almost immediately following ROSC, the patient’s hemodynamics improved to a heart rate 87bpm, respiratory rate of 20, blood pressure 139/67mmHg, and oxygen saturation 99% on the ventilator. She was also following commands and moving all extremities. A computed tomography angiogram (CTA) of the chest showed extensive acute thromboembolic clot burden involving the right and left main, lobar, segmental, and subsegmental arteries .CPC-EM CapsuleWhat do we already know about this clinical entity?Death from massive pulmonary embolism (PE) is due to hemodynamic collapse. The only emergency department intervention that likely improves survival rates in massive PE is thrombolysis.What makes this presentation of disease reportable?Not much is known about the ideal route and dose of tissue plasminogen activator for patients with massive PE who have hemodynamic collapse.What is the major learning point?We suggest a central catheter-directed thrombolytic bolus to improve drug efficacy in patients with cardiac arrest from massive PE.How might this improve emergency medicine practice?This specific treatment modality may reduce mortality rates in patients with massive PE.The patient was admitted to the medical intensive care unit for further management. She was discharged home on hospital day 13 at her baseline functional status on oral apixaban.We present a case of massive PE that was strongly suspected based on history and physical exam and supported by the use of point-of-care ultrasound. The resulting hemodynamic collapse in this elderly female was thought to be successfully treated with CVC-directed tPA. The patient had return of cardiac function, resolution of hypoxia, and ultimately had a favorable outcome. It has been described that patients with massive PE and right ventricular dysfunction have a more rapid return of ventricular function and pulmonary perfusion with a thrombolytic bolus.4For patients with massive PE and hemodynamic instability, poor peripheral perfusion from right sided heart failure may limit tPA efficacy if administered through a peripheral intravenous line. Thus, the decision to bolus tPA through the CVC is hypothesized to increase concentration at the site of the thrombus. To date, there have been no randomized controlled trials that compare central to peripherally dosed thrombolytics. To our knowledge, there has been one published case report that describes CVC-directed thrombolytics, which also had a favorable outcome.Patients in cardiac arrest or peri-arrest in the setting of PE may benefit significantly from a bolus of tissue plasminogen activator through a central venous catheter. In the future, more evidence is needed to analyze CVC-directed tPA and its effect on morbidity and mortality.


Cancer is the leading cause of premature death in Canada. In the last decade, important gains in cancer survival have been achieved by advances in adjuvant treatment. However, many oncologic treatments also result in cardiovascular "toxicity". Furthermore, cardiac risk factors such as hypertension, dyslipidemia, and diabetes mellitus are known to contribute to the progression of cardiac damage and clinical cardiotoxicity. As such, for many survivors, the risk of death from cardiac disease exceeds that of recurrent cancer. While provision of care by multidisciplinary teams has been shown to reduce mortality and hospitalizations among heart failure patients, the effect of assessments and interventions by multidisciplinary specialists in cancer patients receiving cardiotoxic chemotherapy regimens is currently unknown. Accordingly, we will examine the effect of a multi-disciplinary team interventions in the early assessment, identification and treatment of cardiovascular risk factors in cancer patients receiving adjuvant systemic therapy. Our main hypothesis is to determine if the incidence of LV dysfunction in cancer patients undergoing adjuvant therapy can be reduced through a multidisciplinary team approach.This is a randomized study comparing intensive multidisciplinary team intervention to usual care in the prevention of LV remodeling in patients receiving anthracycline or trastuzumab-based chemotherapy. Main objectives include early detection strategies for cardiotoxicity using novel biomarkers that reflect myocardial injury, remodeling and/or dysfunction; early identification and intensive treatment of cardiovascular risk factors; and early intervention with supportive care strategies including nutritional and pharmacist counselling, exercise training and cardiology team support. Secondary objectives include correlation of novel biomarkers to clinical outcomes; correlation of multidisciplinary interventions to adverse clinical outcomes; relationship of multidisciplinary interventions and chemotherapy dose density; preservation of lean muscle mass; and patient reported outcomes .Cardiac toxicity as a result of cancer therapies is now recognized as a significant health problem of increasing prevalence. To our knowledge, TITAN will be the first randomized trial examining the utility of multidisciplinary team care in the prevention of cardiotoxicity. We expect our results to inform comprehensive and holistic care for patients at risk for negative cancer therapy mediated sequelae.NCT01621659 Registration Date 4 June 2012.ClinicalTrials.gov,  As a result of improved anti-cancer therapies, many patients now experience long-term survival after treatment. However, cardiac toxicity of cancer therapy is increasingly recognized as a major risk, such that for many survivors, the risk of death from cardiac disease exceeds that of recurrent cancer . Negativ2, indicating that even conservative doxorubicin dosing conveys long-term cardiac sequelae. These observations herald an emerging and potentially devastating health issue, as 60,000 patients per year are exposed to anthracyclines in the United States alone [The most common manifestation of cardiotoxicity during or after cancer therapy is left ventricular (LV) dysfunction and heart failure (HF) . Cancer es alone . Maximizes alone . Therefopeak) and unfavorable lipid profiles were observed compared to controls [Our group has observed high prevalence and negative influence of cardiovascular risk factors in breast cancer patients. In 41 breast cancer patients who had received trastuzumab-based chemotherapy, we showed that at ~20 months following completion of therapy survivors consistently had higher rates of cardiovascular risk factors. Here, higher rates of overweight/obesity, low cardiorespiratory fitness .TITAN (multidisciplinary Team IntervenTion in cArdio-oNcology) is a randomized study comparing intensive multidisciplinary team intervention to usual care in the prevention of LV remodeling in patients receiving anthracycline or trastuzumab-based chemotherapy. Additional inclusion requirements are shown in Table The TITAN study underwent full Board review and was approved by the University of Alberta Health Research Ethics Board—Biomedical Panel. Potential participants are consecutively identified at Tumor Board review of all newly diagnosed cases of early breast cancer and lymphoma scheduled for chemotherapy (anthracycline and/or trastuzumab-based). Following primary oncologist approval, a study coordinator provides potential participants with an overview of the study and written information. Written informed consent to participate in the TITAN study is obtained from all participants. After informed consent, participants are scheduled for a baseline clinic visit for final determination of eligibility.VO2peak) is evaluated with an incremental exercise test; continuous expired gas analysis is performed with a metabolic measurement system. Heart rate and cuff blood pressure are measured at each stage; the highest oxygen consumed over 1-min will be used as the peak VO2 score [Clinical assessments include physical examination, cardiovascular history, symptomatology and risk factor profile. Smoking status, vital signs, menstrual status, weight and height will acquired. Cardiac MRI, coronary artery calcium score (cardiac CT) and body composition  will be acquired. Maximal aerobic capacity . Each aGroup 1: Multidisciplinary Team Intervention2) will be performed on a cycle ergometer or treadmill. Thereafter, participants will perform moderate intensity contimuous exercise (1 day/week) and high-intensity aerobic interval training. Participants will also perform 1–3 sets of moderate upper  to lower extremity  resistance training (10 to 15 repetitons with weight added after 3 sets are completed while adhering to strict technique). All subjects will be given an exercise diary to record exercises performed, training heart rate, weight lifted and rate of perceived exertion.Patients randomized to the MDT arm will be assessed by the cardio-oncology team, and receive regular clinical assessment. Blood pressure and lipid profile targets will be guided by national guideline consensus documents , 32. CouGroup 2: Usual CarePatients randomized to usual care will receive standard anti-cancer therapy and standard clinical monitoring.The exercise assessments  are performed at a different facility by different staff than the supervised exercise training team. The cardiac MRI interpreter is blinded to the group assignment.The incidence of left ventricular systolic dysfunction in early breast cancer patients receiving trastuzumab has been reported as approximately 10 % in randomized clinical trials  and up tStudy data are collected and managed using REDCap electronic data capture tools hosted at the University of Alberta . REDCap Maximal exercise testing is safe for patients with early and metastatic cancer; the exercise protocol in this study has been used in healthy older subjects, cardiac patients and breast cancer survivors , 25, 26.Cardiac toxicity as a result of cancer therapies is now recognized as a significant health problem of increasing prevalence. Not only deleterious cardiac effects, but negative impacts on the entire oxygen cascade and multiple organ systems highlight the need for multiple interventional approaches as we will undertake here. To our knowledge, TITAN is the first randomized trial examining the effect of multidisciplinary team interventions in the prevention of chemotherapy-related cardiotoxicity. We expect that, in the short-term, this study will find that early multidisciplinary care will maintain cardiac geometry and enhance patient-reported quality of life. In the long-term, we anticipate that early supportive care interventions will prevent deleterious cardiovascular effects of cancer treatment, and improve overall survival (both cancer and cardiovascular). We also expect this work to inform new approaches in the delivery of comprehensive and holistic care in patients at risk for cancer therapy-mediated cardiovascular toxicity.Patients at risk for or with cancer treatment-related cardiac effects are complex, multi-disease patients requiring multiple specialists. The TITAN Study will provide high-level evidence in the development of guidelines for multidisciplinary preventive initiatives, locally, nationally and internationally. Lastly, this work will inform and support other long-term goals of our cardio-oncology team, in the prevention, detection and treatment of cardiovascular effects of cancer therapies.


Accumulating evidence suggests that noncoding RNAs (ncRNAs) have important regulatory functions. However, lacking of functional annotations for ncRNAs hampered us from carrying out the subsequent functional or predictive research. Here we dissected the expression profiles of 3,458 rat noncoding genes using rat bodymap RNA-sequencing data consisting of 11 solid organs over four developmental stages  from both sexes, and conducted a comprehensive analysis of differentially expressed noncoding genes (DEnGs) between various conditions. We then constructed a co-expression network between protein-coding and noncoding genes to infer biological functions of noncoding genes. Modules of interest were linked to online databases including DAVID for functional annotation and pathway analysis. Our results indicated that noncoding genes are functionally enriched through pathways similar to those of protein-coding genes. Terms about development of the immune system were enriched with genes from age-related modules, whereas terms about sexual reproduction were enriched with genes in sex-related modules. We also built connection networks on some significant modules to visualize the interactions and regulatory relationship between protein-coding and noncoding genes. Our study could improve our understanding and facilitate a deeper investigation on organ/age/sex-related regulatory events of noncoding genes, which may lead to a superior preclinical model for drug development and translational medicine. Functional investigations on protein-coding genes have been well studied, whereas the functions of noncoding RNAs (ncRNAs) are rarely understood. Previously, transcribed ncRNAs were simply regarded as accumulated debris from intronic and intergenic regions of the genome during the transcriptional process1348910111213Previous studies indicated that noncoding genes show more sensitivity to organ and age factors compared to protein-coding genes, thus we chose ten different kinds of solid organs from each rat across four ages to study the regulatory changes in rat development15Rat is the second most widely used model animal for biomedical research. It has greatly contributed to our understandings of the mechanisms of diseases and has served as a great tool for safety evaluation of numerous medicines192022252627Here we characterized DEnGs from multiple perspectives on 3,458 noncoding genes among 320 rat RNA-seq samples covering 11 solid organs across four developmental stages for both female and male. We also constructed the co-expression network between noncoding genes and protein-coding genes. Some significant and meaningful modules related to organ, age and sex were revealed. We further annotated these related modules to explore the potential functions of ncRNAs. Our study provides a comprehensive overview of the expression characteristics of ncRNAs in rat.To obtain an overview of the expression profiles of noncoding genes in rat, we first extracted the expression values of 3,458 noncoding genes with expression level FPKM > 0.01 using CufflinksFrom the principal variance component analysis (PVCA) , we founTo explore the development-related DEnGs, we used Cuffdiff for all genes including noncoding ones at both gene and transcript levels. We performed statistical analysis to identify significant DEnGs between any two consecutive developmental stages across 11 organs, and between sexes. The threshold was set as Benjamini-Hochberg adjusted p-value ≤0.05 plus an expression-level change of at least two times higher or lower (fold change ≥2 or ≤0.5). Generally, male rats shared more developmental stage-dependent DEnGs than female except for adrenal gland and heart. The numbers of DEnGs varied from 19  to 503 (testis) across all tissues . Adrenal3132Although sex difference is not as strong as that of organ or age, we set the same threshold of Benjamini-Hochberg adjusted p-value ≤0.05 with the same fold change cutoff as above on the nine non-sex organs from every developmental stage to identify sex-biased noncoding genes . The numi.e. Benjamini-Hochberg adjusted p-value ≤0.05 and a fold change ≥2 between two organs. The numbers of DEnGs turned out to be varied dramatically as different pairs of organs were compared. A relatively larger number of DEnGs was obtained when brain and liver were compared with other organs among all four developmental stages. For brain, its special biological functions required the expression of a large number of unique genes compared with other organs, whereas for liver, the faster metabolism of liver cells may cause the difference. Yet a fewer number was detected when uterus was compared with other organs. However, for testis and thymus, the number of DEnGs was remarkably age dependent. Testis showed the largest numbers of DEnGs at week 6 and week 21 when in comparison to other organs, but the least numbers of DEnGs occurred at week 2 and week 104  distinct gene modules were identified from the 16,346 organ-related genes. The modules were grouped together by similar tissue types or biological systems. First, the blue module  consiste3942To evaluate the function of sex-related noncoding genes, 1,603 sex-related informative genes were clustered into four modules  after dynamic tree cutting . Each moFor the age-related gene co-expression network, 14 stable modules including 3,572 genes were found . The bluWe correlated the eigengenes from 3,572 age-related genes to study the relationships between the identified modules and age . As was An increasingly large number of publications have emphasized the important regulatory roles of long noncoding RNAs20454647Our analysis also showed that most noncoding genes were expressed at lower levels than protein-coding genes. Nevertheless, they still play important roles in post-transcriptional regulation, and their functions could in a way be inferred from co-expression network analysis. Here, we built up functional annotations for noncoding genes related with organ, age and sex by linking noncoding genes and protein-coding genes together. In addition, the co-expression network can further bridge the noncoding genes with biological processes. The genes enriched in co-expression network modules were involved in similar cellular compartments due to the reliable modularityWe also found that the tissue specificity is likely driven by the synergistic effect of multi-functional genes. As different types of cells have different ways of metabolism and cell renewal cycle, they may express differently during various developmental periods. For instance, cells in liver and muscle tissues refresh themselves every several months. In general, the number of DEnGs between 6- and 21-week old was lower than those in any other developmental stages, indicating the maturity of the organs at 6-week. Week 2 and week 6 rats displayed the largest number of DEnGs, which could be attributed to puberty. For old rats (104-week), organs like spleen, testis and thymus begin to experience functional recession or atrophy, which also resulted in a large number of DEnGs compared to a previous development stage (21-week). This was also observed by co-expression analysis in that 32 modules were obtained after merging for organ-related genes, of which the number is far bigger than that of organs. That is, genes in modules perform various fundamental cellular functions such as stress response and cell motility from the nerve system. The modules are clustered by genes performing fundamental functionalities of the nerve and neuron systems. The large size of the module is consistent with the complicated biological neural networks present almost everywhere in the human body.In this study, we provided a useful model for characterizing and annotating rat noncoding genes, covering multi-organs and various development periods in both sexes. To the best of our knowledge, it is the first study to build a comprehensive and reliable platform for monitoring the expression fluctuation of noncoding genes among multiple organs along with aging, which builds the foundation for future research on noncoding gene-expression profiles in rat. Moreover, our results can facilitate understanding on changes of noncoding genes expression during developmental stages, providing us an opportunity to assess the potential functions of ncRNAs associated with each condition during the development of organs. Our work will benefit biomedical research as well as drug development by facilitating comprehensive expression profiling and functional annotation of noncoding genes in rat.et al.’s deposit in GEO (GSE53960). The data set consisted of transcriptome sequencing data  of 320 rat bodymap samples from 32 rats with 10 organs  from each rat. The 32 rats were sacrificed at four different ages  for both sexes  in four biological replicates.The raw reads were downloaded from Ying Raw reads were mapped back to the rat reference genome EnsemblAs we have four biological replicates for each of the 80 different conditions, Cuffnorm was performed among 320 samples to access the significant changes in expression for genes between any two conditions. After the expression level was normalized for each gene, we excluded the protein-coding genes from the expression matrix and 3,458 expressed noncoding genes remained. We applied hierarchical clustering analysis (HCA) to visualize the overall expression profiles. Principal variance component analysis (PVCA) with Rp-value was set as 0.05 for differential expression analysis. For organ dependent DEnGs analysis, we combined male and female rats together in each time point and compared each pair of two different organs (adjusted p ≤ 0.05). Age-related DEnGs were selected by the same method as that of organ-related DEnGs. We only compared the adjacent age points in a time series, and sexes were considered separately. Similarly, sex-related DEnGs were examined across the four developmental stages among ten distinct organs.With the aligned results by TopHat2, we used Cuffdiff, a downstream program within Cufflinks, together with the rat Ensembl Rnor_5.0 GTF file, to conduct noncoding transcriptome assembly and to identify differentially expressed noncoding genes (DEnGs) related to organ, age and sex. The threshold of false discovery rate (FDR) adjusted p ≤ 0.05 to select genes that were potentially functionally and specifically relevant to organ, age and sex. Finally, 16,346 genes were identified as organ-related, 3,572 genes as age-related, and 1,603 genes as sex-related. The weighted gene co-expression network analysis between protein-coding genes and noncoding genes was constructed using WGCNA packageWe first excluded those lowly expressed genes with FPKM < 0.1. Then, we performed an analysis of variance (ANOVA) on all 26,689 rat genes and used Benjamini-Hochberg adjusted Two selection standards were set to meet the criteria. First, we chose modules with more noncoding genes so that their possible biological roles can be inferred by those of the protein-coding genes involved in the same pathway or biological process. Secondly, we set a threshold for each module to control the strength of inter-nodes connectivity. By referring to the eigengene dendrogram that reveals the relationships between modules and factors, one can choose modules that are relatively highly related to a trait (i.e. age). One must make sure that their mutual correlations were stronger than other modules to age, for instance. Finally, we exported the edge file of those genes into Cytoscape v3.2.1 to make a network so as to specify the weighted link among genes.How to cite this article: Wen, Z. et al. Expression profiling and functional annotation of noncoding genes across 11 distinct organs in rat development. Sci. Rep.6, 38575; doi: 10.1038/srep38575 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Psychiatric research is still strongly influenced by its major classification schemes, the DSM-5  and ICD-No single psychiatric diagnosis has reliably shown to represent a discrete taxa, i.e., clearly demarcated disorder entities that reliably delineate disorders from each other (between-disorder boundary) and normality from disease (within-disorder boundary) –10. A spIn the following, we will briefly recapitulate some of the most pressing limitations underlying current nosology using the example of the 10 DSM-5 personality disorder (PD) diagnoses, as experts in PD research have repeatedly emphasized that these diagnostic categories are neither reliable nor valid –29. MoreThe HiTOP was developed by an independent consortium of psychopathology researchers . This prThe RDoC program was initiated by the National Institute of Mental Health (NIMH) due to the organization’s discontent with the current psychiatric classification system . As psycHiTOP and RDoC are recently developed dimensional frameworks aimed at replacing the systematically flawed classification systems of DSM-5 and ICD-10 for research purposes. These two pioneering approaches are best seen as complementary rather than as competing systems. Both approaches have in common that they adopt a hierarchical, fully dimensional scheme. However, while HiTOP focuses exclusively on psychopathological symptoms, syndromes, and broad phenomenological domains, RDoC is largely aimed at examining genetic and neurobiological units of analysis. Therefore, RDoC holds particular promise for advancing neurobiological research relevant to psychopathology, while HiTOP is mainly concerned with restructuring psychiatric nosology by providing an empirical organization of psychopathology . PsychiaMH drafted the manuscript. SL contributed in writing and critical revision.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Parvoviridae) that have recently been identified via metagenomic sampling of animal faeces. Here, we report the sequences of six novel chapparvoviruses identified through both metagenomic sampling of bat tissues and in silico screening of published vertebrate genome assemblies. The novel chapparvoviruses share several distinctive genomic features and group together as a robustly supported monophyletic clade in phylogenetic trees. Our data indicate that chapparvoviruses have a broad host range in vertebrates and a global distribution.Chapparvoviruses are a highly divergent group of parvoviruses (family  Densovirinae, which contains viruses that infect invertebrate hosts, and Parvovirinae, which contains viruses that infect vertebrate hosts. A total of eight genera have been recognized within the subfamily Parvovirinae 13, 14].Parvovirinae. We constructed a multiple sequence alignment spanning 113 residues within this region and containing representative Parvovirinae reference sequences in addition to novel sequences. A combination of automated procedures  and manual adjustment were used to create the final multiple sequence alignment [As shown in lignment , which wlignment . As showParvovirinae (i.e. ~500 amino acids as compared to ~700). Also, the predicted capsid proteins of newly characterized chapparvoviruses contained phospholipase A2 motifs in their N-terminal regions. These motifs, which are reportedly involved in intracellular trafficking and/or escape from endosomes, are found in many, but not all, members of the Parvoviridae [Complete capsid ORFs are present in two previously obtained chapparvovirus genome sequences (rat parvovirus and porcine parvovirus 7) and two that were obtained in our study (DrPV-1 and Cebus capucinus parvovirus). Where complete capsid ORFs are present, they are significantly shorter than found in other members of the subfamily oviridae . Notablyoviridae . PhylogeErythroparvovirus) within the Parvovirinae. The relatively small size of the chapparvovirus capsid protein, combined with the presence of overlap between the capsid and replicase genes, suggests a selection pressure for smaller genome size in these viruses. If we assume that the capsid gene found in these viruses shares a common origin with those found in other Parvovirinae genera, then it appears that this genus has evolved a smaller overall genome size, reducing the size of the capsid gene, while the replicase gene has remained approximately unchanged. Interestingly, this goes against the well-established hypothesis that virus genome size is physically limited by length constraints on genes encoding icosahedral capsids [Parvovirinae genera, an alternative scenario can also be considered wherein the shorter chapparvovirus capsid gene has a separate evolutionary origin to the one found in the other genera.We noted that the replicase and capsid genes of chapparvoviruses often overlap slightly , a trait that has only been observed in one other genus ( capsids . HoweverThe clustering of chapparvovirus sequences into host-class-specific sub-lineages see  is consi


A sequential discrimination–amplification (SEDA) electrochemical strategy was constructed for the identification of single-copy circulating methylated DNA. e.g. plasma), challenging the accurate analysis of specific DNA methylation patterns. Yet conventional assays based on the real-time quantitative methylation-specific PCR (qMSP) are generally limited in detection sensitivity and specificity due to its non-specific amplification interference including primer dimers and off-target amplification. Here we propose a single copy-sensitive electrochemical assay for circulating methylated DNA with ultrahigh specificity on the basis of a sequential discrimination–amplification strategy. Methylated DNA rather than unmethylated DNA in a bisulfite-modified sample is identified and amplified by the asymmetric MSP to generate abundant biotin-labeled single-stranded amplicons with reduced primer–dimer artifacts. Self-assembled tetrahedral DNA probes, which are readily decorated on an electrode surface as nanostructured probes with ordered orientation and well controlled spacing, enable the highly efficient hybridization of the specific single-stranded amplicons due to greatly increased target accessibility and significantly decreased noise. The interfacial hybridization event is quantitatively translated into electrochemical signals utilizing an enzymatic amplification. The proposed assay integrates dual sequence discrimination processes and cascade signal amplification processes, achieving the identification of as few as one methylated DNA molecule in the presence of a 1000-fold excess of unmethylated alleles. Furthermore, the excellent assay performance enables tumor related methylation detection in lung cancer patients with 200 microlitre plasma samples. The results are in good consistency with those of clinical diagnosis, whereas the conventional qMSP failed to detect the corresponding methylation pattern of these clinically confirmed positive patients in such trace amounts of samples.Tumor-related circulating methylated DNA represents only a small fraction of the total DNA in clinical samples ( The methylation of the promoter region CpG islands in tumor suppressor genes is frequently associated with the progression of cancers such as lung cancer.13 Generally, three different strategies including sodium bisulfite conversion,9 restriction enzyme digestion12 and affinity enrichment13 are employed to identify methylated cytosines (mC) from unmethylated cytosines (C). Notably, the bisulfite-assisted methylation-specific PCR (MSP) has been one of the most common methylation analysis tools. This technique relies on the sodium bisulfite treatment of DNA, which converts C to uracils while leaving mC unaffected, thus turning the epigenetic difference into the sequence difference. The modified DNAs are then amplified by the PCR with methylation-specific primers, the products of which are identified via gel electrophoresis. However, this MSP approach offers only qualitative analysis and low assay sensitivity. The real-time quantitative MSP (qMSP)15 takes advantage of the fluorescent dye reporter or TaqMan probe to achieve quantitative analysis, and improves the sensitivity. Nevertheless, it still often suffers from non-specific amplification including primer dimer formation and off-target amplification of unmethylated alleles.16 Besides the real-time qMSP, several novel end-point MSP strategies have been developed to solve these problems.21 Notably, the method  reduced the number of PCR cycles required to inhibit the formation of primer dimers and pushed the detection limit of methylated DNA down to 15 pg.19 However, off-target byproducts from unmethylated DNA amplification still exist and can be detected by FRET as interfering signals.22 Nie and coworkers employed a supercharged green fluorescent protein as a versatile probe for the detection of MSP products, achieving the highly sensitive detection of methylated DNA extracted from human colon carcinoma tissue samples.21 Furthermore, this method used toehold strand displacement hybridization to improve the specificity by single-base mismatch between methylation and unmethylation sequences. In addition, Li’s group has developed an alternative methylation assay based on the ligase chain reaction. This assay evaded the problems of primer dimers and off-target amplification in the MSP, achieving the determination of as low as 10 aM and 0.1% methylated DNA from a large excess of unmethylated DNA with multiplexed methylation sites.23 Yet, mismatch ligation24 and blunt-end ligation also exist in the ligase chain reaction, causing non-specific signals. Thus, developing a highly specific and sensitive methylated DNA detection strategy without non-specific amplifications still remains a challenge.Numerous available tools to assess DNA methylation have been successively developed over the past two decades.25 we speculate that decreasing the concentration of the primers can reduce and even eliminate the dimers, for example, in the asymmetric PCR. However, it conversely suffers from low amplification efficiency. Thus the integration of a downstream amplification strategy is required to further improve the sensitivity.27 Additionally, as off-target amplification still remains in the asymmetric PCR, the combination of specific downstream discrimination can further improve the specificity.31 Recently, we developed a tetrahedral DNA nanostructure-based electrochemical detection strategy.33 These tetrahedrons provide greatly increased target accessibility and significantly minimize non-specific adsorption. By simultaneously utilizing enzymatic amplification, we realized the detection of nucleic acids in the aM-level. Collectively, by integrating the above solutions, we expect to overcome the problems of primer dimer formation and off-target amplification, realizing methylation detection with high sensitivity and high specificity.Considering that the high concentration of the primers is always accompanied by the generation of primer dimers,via decorating self-assembled tetrahedral DNA on a gold electrode surface were employed to capture these amplicons. These probes greatly increased target accessibility and minimized the non-specific adsorption of amplification byproducts due to the rigid scaffold, ordered orientation and well controlled spacing, thus enabling the high efficiency and specific hybridization and significantly decreasing noise. Horseradish peroxidase-conjugated avidin (avidin–HRP) was then captured via biotin–avidin binding to generate an electrochemical signal by the enzyme catalytic process. Dual sequence discrimination processes, including methylation-specific annealing and specific interface hybridization, as well as cascade signal amplification processes represented by the asymmetric MSP and HRP catalytic reaction, were well integrated in the proposed assay, realizing the detection of single-copy methylated alleles. Moreover, the DNA methylation at the p16INK4a gene promoter in trace amounts of plasma samples (200 microlitres) from eleven lung cancer patients was accurately determined by our proposed strategy, the results of which are in good consistency with those of clinical diagnosis.Herein, we developed a sequential discrimination–amplification (SEDA) strategy for circulating methylated DNA detection with single-copy sensitivity and ultrahigh specificity. In this method, methylated DNA rather than unmethylated alleles in a bisulfite-modified genomic sample was identified and amplified by the asymmetric MSP (AMSP) using a biotin-labeled methylation-specific primer, producing lots of biotin-labeled single-stranded amplicons with reduced primer–dimer artifacts. DNA nanostructured probes The design principle of the proposed SEDA strategy is illustrated in 34 using a cationic redox marker, RuHex, we can prove the immobilization of the DNA tetrahedron on the electrode and further calculate the surface density . A definite amount of tetrahedral probe can theoretically hybridize with an equal amount of amplicon. Therefore, 1 μl of the reaction solution may be approximately saturated. To validate this, we further tested 3 μl of the solution and the result in Fig. S2According to the chronocoulometric quantitation method reported,y Fig. S1 to be 4.+ concentration in the hybridization buffer.35 As shown in + in the hybridization system induced rapid background growth, whereas relatively slow signal growth was observed. Therefore, in order to avoid the impact of an excessive background, we chose 200 mM of Na+ for the following experiment. In addition, we tested the hybridization time and concluded that 30 min is enough for the reaction  can be observed when the DNA amount is relatively low. When the DNA amount was increased, an exponential curve was observed, which indicates that more and more tetrahedral probes were occupied and the second amplification of the signal continued. A plateau effect is nearly reached when a common ng-level of DNA was used as the template in the AMSP, which corresponds with the almost saturated probes. Compared to the conventional qMSP which usually has ng-level sensitivity  and unmethylated (U) DNA, where the presence of unmethylated sequences can potentially influence the sensitivity and the specificity of the diagnostic assay.37 Particularly, for early cancer detection, it is essential to discriminate the low amount of methylated DNA from a high background of unmethylated DNA. Therefore, we investigated the ability of our approach to detect the DNA methylation index by mixing artificially methylated DNA with unmethylated DNA  in a total amount of 15 ng (assuming 100% recovery in the conversion process) at different proportions representing 0, 0.1, 0.2, 0.5, 1, 5, 20, 50 and 100%. As shown in i.e. primer dimers) and the exposed gold electrode surface . The inset of R2 = 0.996) was observed, proving that the presence of the unmethylated sequence does not interfere with the target methylated sequence detection in our approach. Meanwhile, the overly excessive unmethylated DNA interferes with the result obtained from the qMSP  of plasma from lung cancer patients. Therefore, our single-copy sensitive electrochemical assay is superior, and we believe that it has an immediate and promising impact on fundamental research and clinical applications.


Immune checkpoint inhibitors (ICPIs) are being investigated in clinical trials for patients with glioblastoma. While these therapies hold great promise, management of the patients receiving such treatment can be complicated due to the challenges in recognizing immune-related adverse events caused by checkpoint inhibitor treatment. Brain imaging changes that are the consequence of an inflammatory response may be misinterpreted as disease progression leading to inappropriate premature cessation of treatment. The aim of this study was to, by way of a series of cases, underscore the challenges in determining the nature of contrast-enhancing masses that develop during the treatment of patients with glioblastoma treated with ICPIs.We reviewed the clinical course and management of 4 patients on ICPIs who developed signs of tumor progression on imaging. These findings were examined in the context of Immunotherapy Response Assessment in Neuro-Oncology (iRANO) guidelines. Although all 4 patients had very similar imaging findings, 2 of the 4 patients were later found to have intense inflammatory changes (pseudoprogression) by pathologic examination.A high index of suspicion for pseudoprogression needs to be maintained when a patient with brain tumor on immunotherapy presents with worsening in an area of a pre-existing tumor or a new lesion in brain. Our findings strongly suggest that pathological diagnosis remains the gold standard for distinguishing tumor progression from pseudoprogression in patients receiving immunotherapy. There is a large unmet need to develop reliable non-invasive imaging diagnostic techniques.ClinicalTrials.gov NCT02311920. Registered 8 December 2014. Despite multimodality treatment approaches and ongoing research, glioblastoma remains a deadly cancer with a median overall survival of 11 to 14.6 months . ClinicaIn order to survive, tumor cells evade the body’s immune system by dysregulating immune checkpoints by the overexpression of immunosuppressive surface ligands , 9. A nuThe use of ICPIs in treatment of advanced tumor is challenging due to the toxicities caused by disinhibition of the immune system, now called immune-related adverse events (irAEs) . AlthougHere we present a series of 4 patients with glioblastoma receiving ICPIs under our ongoing phase I clinical trial of ipilimumab, nivolumab and the combination in patients with newly diagnosed glioblastoma (NCT02311920), who developed a new brain lesion or had a radiographic worsening of pre-existing brain tumor. This clinical trial was approved by the institutional review board. All patients had signed written informed consents for participating in the trial and provided written consents for publication. All 4 patients underwent a gross total resection (GTR) of their primary glioblastoma followed by 6 weeks of radiation with concurrent temozolomide (TMZ) chemotherapy. Patients were enrolled in the study at the 4-week follow-up after chemoradiation and were randomized either to nivolumab or ipilimumab arm. All patients received adjuvant TMZ. The initial diagnosis of glioblastoma was confirmed at the Laboratory of Pathology, NCI Figs. . These cA 51-year-old woman (P1), who was previously healthy, presented with chief complaints of a 4-week period of speech difficulties. Cranial MRI revealed a left frontal mass which was enhanced with gadolinium Fig. , A and aDespite administration of dexamethasone, the brain MRI showed a gradual increase in size of the left frontal lesion with increased contrast enhancement and increased blood perfusion Fig. . WorseniA 63-year-old man (P2) presented with intermittent receptive aphasia and was found to have a contrast-enhancing lesion in the right temporal lobe. He underwent a GTR followed by 6 weeks of concurrent radiation and chemotherapy  presented with headaches, nausea and vomiting. Cranial MRI revealed a necrotic enhancing mass in the right temporal lobe with a slight midline shift and uncal herniation. He underwent a GTR, concurrent radiation and chemotherapy  presented with headaches and expressive aphasia. Cranial MRI revealed an irregular contrast-enhancing mass in the left temporal lobe. After a GTR and concurrent radiation therapy and chemotherapy  appeared after initiation of nivolumab. PD-1 and CTLA-4 inhibition have their own unique mechanisms of action. CTLA-4 has both cell-intrinsic (CTLA-4 on effector cells) and cell-extrinsic (CTLA-4 on T cells) activity and is antigen non-specific in contrast to PD-1 which is primarily antigen-specific and cell-intrinsic . Data frThe unique challenges associated with interpretation of new lesions or worsening of pre-existing lesions in patients getting treated with immunotherapy have led to the development of iRANO guidelines in 2015 . The iRAThe iRANO guidelines anticipate that there will be an increased incidence of pseudoprogression with immunotherapy and provide response (and failure) assessment guidelines for clinical trials and clinical care. However, the pseudoprogression timeframe outlined in these guidelines should be prospectively validated to determine this with better accuracy. Furthermore, as we noted from our patient series, asymptomatic lesions may not always be pseudoprogression and true disease progression may not always be symptomatic. Corticosteroids may be initiated to help reverse the mass associated edema, but it has not been established whether inflammatory lesions are more likely to respond than true progressive tumor. Currently, most clinical trials testing immunotherapy in patients with brain tumors state that if clinical examination is stable, immunotherapy can be continued. Conversely, a large increase in the size of the lesions or clinical deterioration would warrant treatment discontinuation unless a biopsy or craniotomy confirms pseudoprogression. Our findings would support tumor sampling or preferably, resection to help establish the diagnosis.However, the interpretation of the tissue obtained from biopsy or resection may be challenging. Small biopsy samples may be misleading because of sampling error and even with extensive resection, the distinction between recurrent and residual tumor can be challenging. No formal criteria have been established to distinguish active, progressive tumor from residual and biologically inactive tumor. Most pathologists rely on tumor cell density, finding of mitotic figures and Ki-67 immunohistochemical staining demonstrating an elevated proliferative index in tumor cells. The finding of accumulated immune cells, including lymphocytes and macrophages, would be expected with pseudoprogression, but there are no established guidelines to help interpret these results. After obtaining a pathological diagnosis, further longitudinal confirmation with stable MRIs will cement the diagnosis of immunotherapy-related pseudoprogression.A high index of suspicion should be maintained for the possibility of pseudoprogression, when patients with glioblastoma treated with immunotherapy develop a worsening of pre-existing tumor(s) or new brain lesions. Currently, clinical examination or advanced imaging techniques cannot accurately distinguish bona fide tumor progression from pseudoprogression. Until reliable non-invasive diagnostic techniques are developed, a pathologic diagnosis with an emphasis on tumor cell density, mitotic activity, Ki-67 immunohistochemical stains and the presence of immune cells, remains the gold standard for the diagnosis of pseudoprogression. The establishment of a pathological diagnostic criterion of immunotherapy-related pseudoprogression will be valuable.


A concomitant tibial shaft and posterior malleolar fracture is a type of regular compound fracture. The associated posterior malleolar fractures are mostly occult fractures, which often do not show a fracture line on ordinary films, and thus lead to a high rate of misdiagnosis. The aim of the present study was to investigate factors helpful for the pre-operative detection of concomitant posterior ankle fractures using the ipsilateral radiographic tibia and fibula shaft fracture characteristics.One hundred eleven adult patients with tibial shaft fractures were selected using inclusion and exclusion criteria. Pre-operative ankle radiographs and computed tomography (CT) scans were obtained for all patients, and clinical data, including age and gender, were collected. Patients were divided into two groups . Fracture height, fracture length, fracture shape, and Haraguchi type of posterior malleolar fracture were measured on radiographs and CT images, and were compared between the two groups. Multiple logistic regression analysis was performed to identify the factors that significantly contributed to concomitant posterior malleolar fractures. Receiver operating characteristic curves were calculated, and cut-off values were used to predict posterior malleolar fractures on pre-operative imaging measurements.Of the 111 patients with tibial shaft fractures, 42 (37.8%) had a concurrent posterior malleolar fracture. Age, gender and affected side were not significantly different, but tibial fracture location, fracture length, and fibular and tibial fracture shape were significantly different between the two groups. In the multiple logistic analysis, tibial fracture location, fracture length, and tibial fracture shape were shown to be significant factors contributing to posterior malleolar fractures. Receiver operating characteristic curves showed that the status of tibial shaft fractures is closely related to the associated posterior malleolar fracture.Ipsilateral posterior ankle fractures are commonly associated with tibial shaft fractures, especially spiral-type injuries. An analysis of the imaging features of such fractures and evaluation of the diagnostic value of various methods can provide imaging basics for the development of accurate and appropriate treatment options. Posterior malleolus fractures are a common type of ankle fracture with variations ranging from small posterior malleolus avulsion fractures to large displaced fractures. The integrity of the posterior malleolus and the posterior tibiofibular ligaments play an important role in load transfer of ankle joints, the stability of the back talus, and the stability of ankle joint rotation . PosteriThe aim of this study was to determine factors helpful for the pre-operative detection of concomitant posterior ankle fractures using the ipsilateral radiographic tibia and fibula shaft fracture characteristics. It was hypothesized that the morphologic features of tibial shaft fractures contribute to posterior malleolus fractures.This study was approved by the clinical research ethics committee of the First Affiliated Hospital of Nanjing Medical University. All patients with tibial shaft fractures underwent CT examinations between 2012 and 2017; the obtained data was retrospectively analyzed. The exclusion criteria were as follows: fractures primarily involving the knee joint ; fractures primarily involving the ankle joint ; old fractures or fractures due to unhealed fractures; congenital dysplasia; neuromuscular disorders; infections; bone tumors and other diseases possibly altering the normal anatomy of skeletal muscle; bilateral comminuted tibial shaft fractures; and fractures without ipsilateral ankle CT images or fractures with X-ray filming excluding the full length of the tibia and fibula .Demographic data, including age and gender, were recorded. As part of our institution’s standard pre-operative evaluation, all tibial shaft fracture patients have orthogonal tibia radiographs with supplemental radiographic views of the ankle and knee. A CT scan without iodinated contrast of the entire tibia that included the ankle was also obtained to assess for concomitant injuries. All CT scans were performed on a 64-slice scanner utilizing 1.25-mm axial slices .Pre-operative radiographs were independently reviewed by three orthopedic surgeons. X-ray films were preliminarily analyzed according to the location of the tibia and fibula fractures. Tibial shaft fractures were divided into four types . Fibular and tibial fracture location was defined as the midpoint of the fracture line, and the point was calculated as the percentage between the ankle and knee joint lines, where the ankle joint line was 0% and the knee joint line was 100%. Similarly, fibular fracture location was calculated as the percentage between the distal and proximal tips of the fibula. Fracture length was defined as the vertical height between the lowest and highest points of the fracture line of the tibial shaft .Three-dimensional evaluation of posterior malleolus fractures with tibial shaft fractures was performed in cross-sectional, sagittal, and coronal surfaces. If the posterior malleolus fracture involves > 25% of the articular surface or fracture displacement is > 2 mm, fixation of the posterior ankle injury  by surgery is necessary .A technique of mapping fracture lines for a given bone, which has been previously described, was used for this study –7. A briThe inter-observer reliability of the three orthopedic surgeons was evaluated using intra-class correlation coefficients (ICCs). The surgeons measured the radiologic indices independently without having access to the patients’ clinical information and the measurement results of other surgeons. One of the orthopedic surgeons repeated the measurements of the radiologic indices 3 weeks after the observer reliability test to assess the reliability of the intra-observer. Inter- and intra-observer reliability testing was performed on 111 patients. After the reliability test, all flat sheet measurements were done by one of the authors who performed intra-observer reliability testing.p value < 0.05 was considered significant [Descriptive statistics, including the mean, standard deviation, and proportions, were determined. A Kolmogorov-Smirnov test verified the normality of the distribution of continuous variables. Comparisons between the patients with and without concomitant posterior malleolar fractures were performed using a t-test or Mann-Whitney U test and a chi-square test according to the data characteristics. To combine the factors that significantly contributed to predict posterior ankle fractures, multiple logistic regression with the stepwise selection method was used. The sensitivity and specificity of the receiver operating characteristic curve were calculated for each radiographic measurement. All statistics were two-tailed, and a nificant .In this study, a total of 111 patients with tibial shaft fractures met the inclusion and exclusion criteria. Of these patients, 72 were males and 39 were females, with an average age of 46.6 ± 16.1 years. Of the 111 patients with tibial fractures, 42 (37.8%) had ipsilateral posterior ankle fractures. Of 42 ankle joint injuries, 30 were isolated posterior malleolus fractures, 11 were posterior malleolus fractures combined with lateral malleolus fractures, and 1 was a posterior malleolus fractures combined with lateral and medial malleolus fractures. In 42 patients with posterior malleolus fractures, 19 underwent surgical treatment and 23 underwent conservative treatment  larger posterolateral-oblique type fragments (Haraguchi type I); and 2) small posterolateral avulsion type fragments (Haraguchi type III; Fig. p < 0.05; Table Radiographic measurements showed satisfactory inter- and intra-observer reliabilities Table . A compaWe then further analyzed age, gender, mechanism of injury, tibial and fibular fracture location, fracture length, and tibial and fibular fracture shape using multiple logistic regression model, and the results showed that tibial fracture location, fracture length, and tibial fracture shape were significant contributing factors to posterior malleolar fracture injuries in tibial fractures Table .Table 3LThe receiver operating characteristic curves for tibial fracture location and fracture length on radiographs of posterior ankle fractures were also calculated. The cut-off value for tibial fracture location was 40.0%, with a sensitivity of 92.9% and a specificity of 44.9% Fig. . The cutThe present study investigated the factors that could be used to detect posterior malleolar fractures concurrent with tibial shaft fractures pre-operatively and to provide cut-off values based on pre-operative radiographic measurements. Tibial fracture location, fracture length, and tibial fracture shape were significant pre-operative factors that were associated with posterior malleolar fractures. When the tibial fracture location is ≤40% or the fracture length is > 11.4% on a radiograph, posterior malleolar fractures should be suspected, appropriately evaluated, and managed at the time of tibial shaft fracture surgery.The incidence of tibial shaft fractures combined with posterior malleolus fractures in each region show remarkable differences. Kempegowda  identifiThe misdiagnosis of tibial shaft fractures combined with ankle fractures reflects the methods of examination. The lack of understanding of this concomitant injury is also an important reason resulting in misdiagnosis. At the time of initial consultation, most orthopedic surgeons only noticed a significant shift in the tibial shaft fracture, but often did not take into account the possible concomitant ankle fracture. When X-ray films were obtained, the ankle joint was not included, and the ankle fracture was therefore missed. Even if most X-ray examinations include the distal tibia, shooting angle and clarity also largely affect the diagnosis of fractures. It is challenging to visualize tibial ankle fractures from frontal films, and it is even more challenging to view the fractures from lateral radiographs due to the overlap with the fibula, especially when the fracture is combined with distal fibula fractures. In addition, most ankle fractures of this type are occult fractures, and it is difficult to diagnose these fractures through X-ray examinations. In these cases, non-displaced distal ankle fractures are often misdiagnosed .Tibial shaft fractures combined with posterior malleolus fractures are a regular combined injury, and the cause of injury, mechanism of injury, and epidemiologic characteristics are different from ankle fractures. It has been shown that the incidence of posterior malleolus fractures in tibial shaft fractures combined with ankle joint injuries is significantly higher than in patients with ankle fractures only  .Ankle fAmong the 42 posterior malleolus fractures in this study, 19 patients underwent surgical treatment and 23 patients were conservatively treated. Surgical fixation is necessary if the posterior ankle fracture is > 25% of the tibial articular surface or the fracture displacement is > 2 mm  found thThe limitations of the current study include those inherent to the retrospective design. Secondly, intra-observer reliability is better to be calculated from the data of the 3 orthopedics. In addition, in our study all tibia shaft fracture surgery was used an intramedullary nail fixation, but because posterior malleolar fractures were performed by different surgeons and different surgical approaches were adopted, including hollow screws and buttress plates, we cannot discuss the choice of surgical methods, and we cannot evaluate functional outcomes after fixation of posterior malleolus fractures with a tibial shaft fracture. Boraiah et al.  reportedOur clinical experience shows that it is necessary to have pre-operative regular radiography, including frontal and lateral X-ray films of ankle joints and carefully check the films, which are critical the diagnosis. For the suspected cases, it is important to perform a CT examination to determine whether or not an ankle fracture is present. In the current study, we performed CT examinations for all diagnosed and suspected tibial shaft fractures with ankle fractures. For the regions that lack medical resources or with high medical costs, diagnosis according to the pre-operative films can save a substantial amount of medical costs. For combined ankle fractures with tibial shaft fractures in busy trauma centers, if ipsilateral ankle joint CT films are not obtained, surgeons can also determine concomitant ankle fracture according to analysis of characteristics in flat films, which can help intra-operative restoration and avoid misdiagnosis.


Pseudogymnoascus destructans load. Increasing fungal load mimicked progression of skin infection from epidermal surface colonisation to deep dermal invasion. Similarly, the number of UV-fluorescent lesions increased with increasing weighted cumulative WNS pathology score, demonstrating congruence between WNS-associated tissue damage and extent of UV fluorescence. In a case report, we demonstrated that UV-fluorescence disappears within two weeks of euthermy. Change in fluorescence was coupled with a reduction in weighted cumulative WNS pathology score, whereby both methods lost diagnostic utility. While weighted cumulative WNS pathology scores were greater in the Nearctic than Palearctic, values for Nearctic bats were within the range of those for Palearctic species. Accumulation of wing damage probably influences mortality in affected bats, as demonstrated by a fatal case of Myotis daubentonii with natural WNS infection and healing in Myotis myotis. The proposed semi-quantitative pathology score provided good agreement between experienced raters, showing it to be a powerful and widely applicable tool for defining WNS severity.While white-nose syndrome (WNS) has decimated hibernating bat populations in the Nearctic, species from the Palearctic appear to cope better with the fungal skin infection causing WNS. This has encouraged multiple hypotheses on the mechanisms leading to differential survival of species exposed to the same pathogen. To facilitate intercontinental comparisons, we proposed a novel pathogenesis-based grading scheme consistent with WNS diagnosis histopathology criteria. UV light-guided collection was used to obtain single biopsies from Nearctic and Palearctic bat wing membranes non-lethally. The proposed scheme scores eleven grades associated with WNS on histopathology. Given weights reflective of grade severity, the sum of findings from an individual results in weighted cumulative WNS pathology score. The probability of finding fungal skin colonisation and single, multiple or confluent cupping erosions increased with increase in  Pseudogymnoascus destructans , , , , }) with an overall Fleiss’ κ5 of 0.78 , signifying good agreement in scoring WNS severity. Inter-rater agreement evaluating each WNS pathology grade separately differed, with basement membrane breach and inflammation being most difficult to score consistently  and lower within-group agreement .Paired scoring agreement corrected for chance between five experienced pathologists and results presented herein ranged from 0.76 to 0.85  Rescue Centre. A time series of UV transillumination and daylight photography images spanning seven days from capture to death indicated that the wing membranes started to show signs of dry necrosis within two days , indicating that single cupping erosions occur in response to roughly one-order-of-magnitude higher fungal loads than that during fungal skin-surface colonisation . Investigating both Palearctic and Nearctic bats  and single cupping erosions  on histopathology dependent on the number of UV fluorescent lesions  than fungal load.Logistic regression  revealednisation . In Paletic bats , a simil lesions . The numβ0 = 62.1, β1 = 16.41; Cumulative WNS pathology scores from single 4 mm biopsies increased with the number of UV fluorescent lesions observed on the surface of the whole wing  and the number of taxa (n = 15) from the Palearctic and Nearctic regions examined, together with the non-lethal nature of the methodology, make this grading system universally applicable to all species exposed to the infection. The ability to select a biopsy site on the bat’s wing displaying the most representative and extensive UV fluorescence [Direct comparison between histological WNS severity scoring from the whole wing membrane, which provides a reasonable representation of skin damage associated with nfection ,20, and rescence  enables P. destructans skin infection in this alternative grading system include presence of single, multiple and/or confluent cupping erosions within a 4 mm punch biopsy that can be evaluated through image analysis to address invasiveness and size [P. destructans to invade the dermis and the full thickness of the wing membrane distinguishes WNS from other dermatophytic infections.Previous WNS grading schemes have proposed four or five WNS-positive grades ,20, withand size , then apand size . Expandiand size ,13. In aAs dysregulated immunity may contribute to severe post-emergent pathology , we alsoP. destructans infection, and the necessity to repair the damaged wing membrane, represent physiological costs that may affect the bat’s fitness and survival. To address the series of healing from different grades of skin damage associated with WNS, our histopathology findings in conjunction with the case reports suggest that cupping erosions heal through marked neutrophilic inflammation Click here for additional data file.S2 Table10 scale per cm2 of wing area. Histopathology is reported as a binary code for index signs of grades corresponding to those listed in Methods and Fungal load and UV fluorescence are given on log(TXT)Click here for additional data file.


Surgery, a crucial therapeutic modality in the treatment of solid tumors, can induce sterile inflammatory processes which can result in metastatic progression. Liver ischemia and reperfusion (I/R) injury, an inevitable consequence of hepatic resection of metastases, has been shown to foster hepatic capture of circulating cancer cells and accelerate metastatic growth. Efforts to reduce these negative consequences have not been thoroughly investigated. Drag reducing polymers (DRPs) are blood-soluble macromolecules that can, in nanomolar concentrations, increase tissue perfusion, decrease vascular resistance and decrease near-wall microvascular concentration of neutrophils and platelets thereby possibly reducing the inflammatory microenvironment. We hypothesize that DRP can potentially be used to ameliorate metastatic capture of tumor cells and tumor growth within the I/R liver.Experiments were performed utilizing a segmental ischemia model of mice livers. Five days prior or immediately prior to ischemia, murine colon adenocarcinoma cells (MC38) were injected into the spleen. DRP (polyethylene oxide) or a control of low-molecular-weight polyethylene glycol without drag reducing properties were administered intraperitoneally at the onset of reperfusion.After three weeks from I/R, we observed that liver I/R resulted in an increased ability to capture and foster growth of circulating tumor cells; in addition, the growth of pre-existing micrometastases was accelerated three weeks later. These effects were significantly curtailed when mice were treated with DRPs at the time of I/R. Mechanistic investigations in vivo indicated that DRPs protected the livers from I/R injury as evidenced by significant decreases in hepatocellular damage, neutrophil recruitment into the liver, formation of neutrophil extracellular traps, deposition of platelets, formation of microthrombi within the liver sinusoids and release of inflammatory cytokines.DRPs significantly attenuated metastatic tumor development and growth. DRPs warrant further investigation as a potential treatment for liver I/R injury in the clinical setting to improve cancer-specific outcomes. Surgery is a crucial intervention which provides a chance of cure for patients with solid tumors. Although surgical excision of primary and metastatic tumors can save or extend life, it has long been acknowledged that the surgical insult itself may precipitate or accelerate tumor recurrence . SterileWhen feasible, resection of hepatic colorectal metastases promotes improved survival compared with chemotherapy alone . UnfortuExperimental studies have elucidated some of the dominant molecular pathways important in the pathogenesis of liver I/R and their role in fostering both the capture of circulating tumor cells and their metastatic growth . Neutropth century, Toms described the ability of small concentrations of soluble macromolecules DRPs to reduce resistance to turbulent flow in a pipe [Drag Reducing Polymers (DRPs) are long-chain, viscoelastic, blood soluble macromolecules with molecular weight over one million Da and a relatively linear structure. In the mid-20n a pipe . DRPs han a pipe . Recentln a pipe . DRPs han a pipe –27. Circn a pipe , 28. Thin a pipe . Since bMale wild-type (C57BL/6) mice (8-10 weeks-old) were purchased from Jackson Laboratories. Animal protocols were approved by the Animal Care and Use Committee of the University of Pittsburgh.DRP (Polyethylene oxide (PEO), 4 million Da MW, Sigma-Aldrich®) was given to animals intraperitoneally at various concentrations. Each polymer preparation was tested for drag reducing and rheological properties as previously described . ControlA nonlethal model of segmental (70%) hepatic warm ischemia and reperfusion was used , 29. DRP4 MC38 cells  were injected into the spleen using a 27-gauge needle. At the same time, mice received either DRP or PEG intraperitoneally. Tumor cells were allowed to circulate for 10 minutes before splenectomy. The next set of experiments was designed to evaluate whether DRP protects from I/R-induced increased growth of already established micrometastatic disease. In these experiments, tumor cells were injected into the spleen through a small left lateral flank incision followed by splenectomy. Micrometastases were allowed to develop throughout the liver for 5 days. Then the mice underwent either hepatic I/R with DRP or PEG injection as described above.The first set of metastases experiments were designed to evaluate whether I/R increased tumor growth in a model of circulating micrometastases. Colorectal liver metastases were induced in mice as previously described . In briet-test. A p < 0.05 was considered statistically significant.Results are expressed as the mean ± standard error of mean (SEM). Group comparisons were performed using ANOVA and Student's We first sought to evaluate the role of different concentrations of administered DRPs on global liver injury after 6 hours of reperfusion. DRPs significantly reduced liver damage as evidenced by reduced serum ALT levels compared with mice receiving PEG in doses as low as 25 ppm and as high as 500ppm; ALT levels were not significantly reduced at 1000 ppm Figure . A DRP cWe have previously shown that liver I/R induced increases in ALT and liver necrosis accompanied by increases in hepatic neutrophil infiltration and intrahepatic NET formation . We therExamination of liver sections of hepatic lobes 6 hours after I/R demonstrated a significant increase in platelet aggregation within liver sinusoids and a significant increase in fibrin-rich microthrombi formation in mice treated with control PEG compared to DRPs Figure  and 3B. p < 0.001). Administration of DRPs after liver I/R resulted in a 69% reduction in tumor nodules compared with untreated I/R mice at 3 weeks   Figure .We have previously shown that established micrometastatic tumors manifest accelerated growth in liver lobes exposed to I/R, an effect partially reversed by inhibition of inflammation associated with NET formation . We usedDuring the surgical resection of metastatic cancer in the liver, some degree of liver I/R injury is inevitable and is associated with worse short term and long term outcomes . We origIndeed, a single intraperitoneal injection of DRP reduced both the number of and bulk of metastatic colon cancer foci resulting from tumor cells injected at the time of liver I/R, but also tumors arising from tumor cells injected 5 days prior to I/R. Coincident with the inhibition of tumor growth in these models, the inflammatory changes induced by I/R was profoundly depressed. There was less liver cell damage, less necrosis, suppressed levels of inflammatory cytokines, less neutrophil infiltration, sinusoidal thrombosis and NET formation. Taken together, the protumorigenic microenvironment was suppressed.in vivo effects of DRPs are incompletely understood, they act primarily in the microvasculature to reduce microturbulence at bifurcations and significantly increase near-wall blood flow velocity [Although the velocity . As a covelocity –36. As aFurthermore, when circulating tumor cells are present, DRPs would be expected to reduce the interactions between tumor cells, platelets and neutrophils by mixing them with the main RBC flow. Since bonds between tumor cells, platelets and neutrophils are known to actively facilitate the metastatic process , 37, DRPThe role of DRPs in reducing neutrophil diapedesis into I/R tissue reinforces the known importance of NET formation as a critical step in the regulation of tumor growth. NETs are important in various infectious and sterile inflammatory processes , 42, 43.in vivo so that the rheological effects are lost within a few days without evident toxicity. Therefore, this is the first study to implicate a role for DRPs as significant therapeutic agent to target the perioperative window to halt the progression of metastases initiated by surgical trauma.These studies were prompted by the clinical problem of increased tumor growth in livers manipulated and thus rendered temporarily ischemic during surgery for cancer. For patients with metastatic or primary liver cancer, liver resection is the primary, though imperfect, means of control. Surgery inevitably creates areas of liver ischemia and reperfusion. It has long been recognized that surgical removal of malignancies may enhance the risk of tumor recurrence . There aIn our experiments, DRPs exhibit an anti-metastatic effect by the combined effects of several possible mechanisms: by improving oxygenation, attenuating pro-tumorigenic pro-angiogenic cytokine interactions, diminish NET formation and decrease microthrombi.In conclusion, this study demonstrates the novel finding that acute intraperitoneal administration of minute concentrations of DRP to mice significantly protected the liver from I/R injury. In addition, DRPs significantly inhibited I/R-induced promotion of metastases development and growth. The clinical implications of our findings are significant as DRPs may offer a powerful novel approach to the treatment of liver I/R injury and perioperative administration of DRPs could improve both short postoperative and long oncologic outcomes.


Recent neuroimaging studies suggest that neutral infant faces compared to neutral adult faces elicit greater activity in brain areas associated with face processing, attention, empathic response, reward, and movement. However, whether infant facial expressions evoke larger brain responses than adult facial expressions remains unclear. Here, we performed event-related functional magnetic resonance imaging in nulliparous women while they were presented with images of matched unfamiliar infant and adult facial expressions  in a pseudo-randomized order. We found that the bilateral fusiform and right lingual gyrus were overall more activated during the presentation of infant facial expressions compared to adult facial expressions. Uncomfortable infant faces compared to sad adult faces evoked greater activation in the bilateral fusiform gyrus, precentral gyrus, postcentral gyrus, posterior cingulate cortex-thalamus, and precuneus. Neutral infant faces activated larger brain responses in the left fusiform gyrus compared to neutral adult faces. Happy infant faces compared to happy adult faces elicited larger responses in areas of the brain associated with emotion and reward processing using a more liberal threshold of p < 0.005 uncorrected. Furthermore, the level of the test subjects’ Interest-In-Infants was positively associated with the intensity of right fusiform gyrus response to infant faces and uncomfortable infant faces compared to sad adult faces. In addition, the Perspective Taking subscale score on the Interpersonal Reactivity Index-Chinese was significantly correlated with precuneus activity during uncomfortable infant faces compared to sad adult faces. Our findings suggest that regional brain areas may bias cognitive and emotional responses to infant facial expressions compared to adult facial expressions among nulliparous women, and this bias may be modulated by individual differences in Interest-In-Infants and perspective taking ability. Baby schema serves as an “innate releasing mechanism”, a fundamental social instinct that serves to initiate and maintain a parent-infant relationship . A numbeInfant facial expressions convey salient information about their emotional states and needs. Accurate interpretation and sensitive caregiving response to the needs underlying infant facial expressions may be critical to infant development . PechtelIt is interesting to note that neural responses in mothers are specific to the processing of infant facial expressions rather than a more generalized bias to any emotional expressions. Specifically, in an event-related potential (ERP) study, early latency of the N1 response was observed for strong intensity facial expressions compared to mild intensity facial expressions, which reflected faster emotional recognition performance. In addition, the early posterior negativity (EPN) waveform is further enhanced by negative infant facial expressions. Neither of these effects is significant in ERPs elicited by adult facial expressions . Using nThe objective of the present study was to explore whether brain response is biased toward unfamiliar infant faces compared to unfamiliar adult faces in nulliparous women, which could further inform our understanding of the neural mechanisms underlying women’s internal processing of caregiving behavior. Previous fMRI studies have shown that happy adult facial expressions activate the basal ganglia, including the ventral striatum and putamen –15, wherWe also examined whether individual differences in Interest-In-Infants and empathy were associated with different patterns of brain response to infant versus adult facial expressions. Freeman  recentlyThirty-five native Chinese-speaking right-handed nulliparous women gave informed written consent and participated in this study, which was approved by the Ethics Committee of Southwest University (NO. 2014179). All research was conducted in accordance with the Declaration of Helsinki. Three subjects exhibited excessive movement during multiple fMRI runs and were excluded from analyses. The remaining 32 subjects were between the ages of 18 and 25 years old , in good health with no history of psychiatric or neurological disorders, and had normal or corrected-to-normal vision.All subjects completed the Interpersonal Reactivity Index-Chinese (IRI-C) , which wInterest-In-Infants  was assePhotographs of infant faces (ages 3–6 months old) were adapted from the Chinese Infant Affective Face Picture System, which was established by our group . All infSubjects viewed 60 intensity-matched face images (ten images for each condition of interest) on each of four functional runs. Images were pseudo-randomized within and between each run but not between subjects. A probe stimulus (a gray image), which subjects were instructed to respond to with a button press, was presented 5 times during each run to maintain the subject’s attention during the task; data from these stimuli were not included in data analyses. All images (face images and probe stimuli) were displayed foveally at the fixation point for 2 seconds with a random inter-stimulus interval of 2, 4, or 6 seconds . Before 3).Functional images were acquired from a 3T Siemens Magnetom Trio Tim B17 MRI scanner . T2*-weighted gradient echo planar imaging sequences were used to obtain functional images. A total of 788 volumes were collected from each subject. Each volume included 33 axial and interleaved acquired slices with 3-mm thickness and 0.6-mm gap oriented parallel to the anterior commissure-posterior commissure (AC-PC) plane . High-resolution T1-weighted images, which were composed of 176 volumes, were acquired for each subject for anatomical reference  in MATLAB version 7.6 . Functional images were realigned for head movement using the median image as a reference, co-registered to the high-resolution structural image, and normalized to the T1 template in the Montreal Neurological Institute (MNI) space. Functional images were resampled to a voxel size of 3 x 3 x 3 mm3 and smoothed with a 4-mm full width at half maximum (FWHM) Gaussian kernel.Data analyses were performed with Statistical Parametric Mapping version 8  from our experiment were modeled as separate regressors using a general linear model. Six head-motion parameters were modeled as independent covariates. All regressors were convolved by a canonical hemodynamic response function, and functional data were high-pass filtered to eliminate low-frequency noise. Six t-contrasts that were created  to generate six statistical parametric maps for each condition. These parametric maps were subsequently submitted to a group-level random-effects analysis.Group-level analyses tested whether emotion type modulates neural activations associated with face type. We entered all six statistical parametric maps of each condition from each subject into a full two-factorial repeated-measures ANOVA that included two within-subjects factors (face type and emotion type). Results for main effects were thresholded at a peak-level family-wise error (FWE) corrected p < 0.05 (voxel-wise uncorrected p < 0.001). Results for the interaction effect were thresholded at a peak-level false discovery rate (FDR) corrected p < 0.05 (voxel-wise uncorrected p < 0.001) with an extent threshold of k = 10.Simple effects were examined using t-contrasts with thresholds at a cluster-level FWE corrected p < 0.05 (voxel-wise uncorrected p < 0.001) for the uncomfortable and neutral face comparisons, but a more liberal threshold voxel-wise uncorrected p < 0.005 was applied to the comparison of happy infant faces to happy adult faces.For clusters exhibiting significant results, we created regions of interest  centered on each peak coordinate , and extracted individual mean beta values of each ROI using MarsBar software . Scores To test our hypotheses, Pearson correlations were calculated between the bilateral FFG beta values and the Interest-In-Infants test score, and between the PCU beta values and IRI-C Likert scores. Then, a multiple regression analysis was conducted to determine which IRI-C factors predict the beta values of the PCU. All predictor variables were added simultaneously using the enter method.The matched intensity ratings were as follows: sad/uncomfortable ; neutral ; and happy . A between-subjects analysis of variance (ANOVA) of the intensity ratings demonstrated that the main effect of emotion (happy vs sad) was significant , but that the main effect of face type  and the interaction between emotion type and face type  did not reach significance.The arousal of faces was rated by an independent group on a scale of 1 (the lowest) to 9 (the highest). A between-subjects ANOVA of the arousal ratings demonstrated that the main effects of emotion types , face types , and the interaction effect  all reached statistical significance. The multiple comparison of emotion types revealed significant arousal difference between happy and neutral facial expressions , happy and sad facial expressions  and sad and neutral facial expressions . The simple effect test of interaction showed significant arousal differences between uncomfortable infant and sad adult expressions  and neutral infant and adult facial expressions , but not between happy infant and adult facial expressions .Group analysis revealed a significant main effect of face type. Activation of the bilateral cuneus was higher for adult faces than infant faces, whereas activation of the bilateral fusiform and right lingual gyrus was higher for infant faces than adult faces ; Fig 2A.Several regions demonstrated an interaction between face and emotion types, indicating that sensitivity to face type was modulated by emotion type in these regions. These regions included the right middle occipital gyrus, with a cluster extending to the right FFG , PCU , PCC , and left THA  ; Fig 3A.2 = 0.044, F  = 1.33, P = 0.285. While this model did not reach statistical significance; perspective taking was the only variable that approached significance, ß = 0.388, t = 1.99, p = 0.057.We found that Interest-In-Infants was positively associated with the beta values of right FFG response to happy, neutral, and uncomfortable infant facial expression. It also associated with the contrast beta values of right FFG response to uncomfortable infant facial expression versus sad adult facial expression , Fig 5. The current study used fMRI to explore whether neural response is biased toward infant facial expressions compared to adult facial expressions in nulliparous women. We found that the bilateral FFG and right lingual gyrus were more activated during perception of infant faces than adult faces. We also observed regional brain response bias to uncomfortable and happy infant faces compared to sad and happy adult faces respectively. The right FFG responses to infant faces and the comparison between uncomfortable infant faces and sad adult faces were positively correlated with Interest-In-Infants. Furthermore, the PCU response bias to uncomfortable infant faces was positively correlated with Perspective Taking ability.Greater activity in the FFG was observed when test subjects viewed infant faces compared to adult faces. Our results are consistent with previous studies, which demonstrated that the FFG plays an important role in encoding the specificity of infant faces , 30–32. The contrast beta values of the right FFG were larger when comparing sad faces than for neutral and happy faces comparisons. In addition, the association between Interest-In-Infants and the contrast beta values of right FFG response to the neutral and happy faces comparisons did not reach significance. These results may be caused by no arousal difference between happy infant and adult facial expressions in our experimental stimuli, and uncomfortable infant facial expressions likely convey more biological meaning than the neutral expressions. The reasons for these results should be further investigated in future studies. The association between the left FFG activity and Interest-In-Infants was not significant, which may be caused by the differential function of the left and right FFG in face recognition .We found that uncomfortable infant faces compared to sad adult faces elicited greater activation of the PCU than comparisons of happy or neutral infant versus adult faces. Consistent with our prediction, the activity in the PCU was significantly correlated with the IRI-C perspective taking factor score (cognitive empathy), but not with emotional aspects of empathy. It is known that the neuroanatomical bases of emotional and cognitive empathy are different. Specifically, the core anatomical structure associated with emotional empathy is the inferior frontal gyrus, which is implicated in emotional contagion and emotion recognition; whereas the neural bases of cognitive empathy are the ventromedial prefrontal cortex, superior temporal sulcus, temporo-parietal junction, and PCU, which are implicated in mentalizing or ToM , 35. ForThe difference in PCC-THA responses observed in this study among sad, neutral, and happy faces is consistent with findings from other studies, in which greater activation in the PCC-THA was observed when mothers listened to infant cries compared to white noise  or when Using a more liberal threshold, we found brain response differences in the OFC, superior frontal gyrus, ACC, and insula cortex when comparing subject responses to happy infant faces and happy adult faces. Two previous studies also reported that mothers exhibited activation of the OFC while viewing happy/positive facial expressions of their own versus unfamiliar infants , 46. TheIn the present study, unfamiliar facial expressions were used to avoid the confound of familiarity. Furthermore, the comparison of infant to adult faces was designed to extract physical features specific to infant faces. Lastly, exploring brain response to infant facial expressions among nulliparous women can help to characterize shifts in maternal brain function. However, there are some limitations to our study. Firstly, the adult face images in our experimental materials were cropped such that the forehead, which is also involved in expressing emotion, was invisible. These images will be modified to include the forehead in a future study. Secondly, some factors which may confound our results should be controlled in a future study, such as the sexual preference in the processing of face perception.In order to determine whether the brain response bias is caused by the distinctive features of the infant face, future studies should control for the arousal level of infant and adult facial expressions. In addition, brain response to infant cues is affected by other factors, such as sex, parental status, or individual differences in parenting motivation and personality traits. For example, brain areas involved in mind wandering are modulated by sex in response to infant cries . We haveIn conclusion, the present study provides an examination of whether the neural response is bias toward infant facial expressions compared to adult facial expressions in nulliparous women. Our findings demonstrated that uncomfortable infant faces increase activation in regions associated with face and empathic processing compared to sad adult faces, whereas happy infant faces enhance activation in regions involved in emotion and reward processing as compared to happy adult faces using a more liberal threshold. Our findings suggest that regional brain areas associated with nulliparous women’s cognitive and emotional responses are biased toward infant facial expressions, which may be consistent with the hypothesis that the caregiving system of nulliparous women matures in the late adolescence period. Furthermore, the brain response bias to infant faces is modulated by individual differences in Interest-In-Infants and perspective taking ability.


Although oncologic testicular sperm extraction (onco-TESE) has been increasingly practiced, the evidence of onco-TESE performed in patients with testicular cancer is insufficient. Furthermore, in bilateral testicular cancer, accounting for 0.5%–1% of testicular cancers, onco-TESE is more challenging and has been insufficiently reported.Here we report the case of a 25-year-old man who underwent onco-TESE from his residual single testis with a nonseminomatous germ cell tumor that occurred 5 years after orchiectomy of the contralateral testis. A second orchiectomy and simultaneous TESE from the noncancerous testicular tissue were performed. The pathological diagnosis was germ cell tumors, tumors of more than one histological type . The patient subsequently married and hoped for fatherhood 3 years later. Whereas histological diagnosis of the normal testicular tissue was Johnsen score 6 (maturation arrest), morphologically normal and motile sperms were successfully retrieved from thawed TESE samples and used for multiple cycles of intracytoplasmic sperm injection. Although the conception has not been succeeded to date, ICSI attempts have been continuing.This case demonstrates the effectiveness of onco-TESE for challenging cases such as bilateral and nonseminmatous testicular cancer. Although oncologic testicular sperm extraction (onco-TESE) has been increasingly practiced, the evidence of onco-TESE performed in patients with testicular cancer, especially bilateral testicular cancer, is insufficient. We present a challenging case of onco-TESE from a single cancerous testis with a metachronous bilateral testicular tumor that was detected at 5 years after orchiectomy of the contralateral testis; this procedure resulted in the successful retrieval of normal sperms, which were used for intracytoplasmic sperm injection (ICSI).A 25-year-old unmarried Japanese man presented to our hospital with a mass in his left testis. He underwent orchiectomy of his right testis 5 years earlier, and histopathological diagnosis revealed immature teratoma of the testis. No salvage chemotherapy was performed. He discontinued follow up several years after the surgery on account of moving away. Five years after the orchiectomy, his residual left testis was noted to be slightly enlarged, and a nodule was palpable. The volume of normal testicular was not measurable due to replacement by the tumor. Ultrasonography revealed a mass with a heteroechoic pattern and 4-cm diameter Fig. . LaboratAlthough testicular cancer is one of the most common malignancies in men of reproductive age , the treThe methodology for onco-TESE in testicular cancer patients has not been established. Onco-TESE for testicular cancer patients is often challenging because the sample needs to be retrieved from noncancerous tissue that has been separated from cancerous tissue. The evidence of using sperms from noncancerous tissue in cancerous testes has not been sufficiently accumulated. To our knowledge, only 27 cases of onco-TESE in testicular cancer patients have been reported in the literature , 6–11. TIt is noteworthy that the normal sperms were successfully harvested and used for multiple ICSI cycles in this case, because the outcomes of onco-TESE, including the success rates of sperm retrieval, use, conception, and pregnancy, have not been sufficiently documented. Among the reported cases of onco-TESE, the success rate of sperm retrieval was 45% in the azoospermia patents with any type of cancer and 59% 16 of 27 cases) in those with testicular cancer , 6–11. T cases inThe safety of using sperms retrieved from cancerous testis in terms of fetal health also remains unclear. Only a few cases have reported birth of healthy baby with onco-TESE from cancerous testis, including one case with bilateral testicular cancer –10. AlthThe present case also suggested the possibility of sperm retrieval with onco-TESE despite unfavorable Johnsen score. Although the Johnsen score in this case was 6, indicating maturation arrest, normal sperms were obtained from the cryo-thawed testicular tissue. This discrepancy occurs possibly because the onco-TESE was performed with the macroscopic detection of normal testicular tissue. Conversely, microdissection TESE (MD-TESE) that enables sampling from microscopically healthier seminiferous tubules may be recommended for onco-TESE. Additionally, this case also showed an inconsistent result between histological and clinical information: the wide intraseminiferous lumen suggesting obstruction of the tubules in contrast to the clinical diagnosis of non-obstructive azoospermia with the elevated sexual hormones. Although it is difficult to clarify the reason for this inconsistency, this case may imply that such dilated intraseminiferous lumen is caused by partial obstruction of the tubules in the microenvironment of cancerous testis in azoospermic patients with testicular cancer.In conclusion, our case presented that motile normal sperms were successfully retrieved from preserved testicular tissue and used for multiple ICSI attempts. Although successful pregnancy has not yet been achieved in this case, the successful experience of sperm retrieval from a residual single testis in a patient with bilateral and metachronous nonseminoma implies the effectiveness of onco-TESE in challenging cases.