PET Imaging of Neurofibromatosis Type 1 with a Fluorine-18 Labeled Tryptophan Radiotracer.

Neurofibromatosis type 1 (NF1) is a neurocutaneous disorder. Plexiform neurofibromas (PNFs) are benign tumors commonly formed in patients with NF1. PNFs have a high incidence of developing into malignant peripheral nerve sheath tumors (MPNSTs) with a 5-year survival rate of only 30%. Therefore, the accurate diagnosis and differentiation of MPNSTs from benign PNFs are critical to patient management. We studied a fluorine-18 labeled tryptophan positron emission tomography (PET) radiotracer, 1-(2-[18F]fluoroethyl)-L-tryptophan (L-[18F]FETrp), to detect NF1-associated tumors in an animal model. An ex vivo biodistribution study of L-[18F]FETrp showed a similar tracer distribution and kinetics between the wild-type and triple mutant mice with the highest uptake in the pancreas. Bone uptake was stable. Brain uptake was low during the 90-min uptake period. Static PET imaging at 60 min post-injection showed L-[18F]FETrp had a comparable tumor uptake with [18F]fluorodeoxyglucose (FDG). However, L-[18F]FETrp showed a significantly higher tumor-to-brain ratio than FDG (n = 4, p < 0.05). Sixty-minute-long dynamic PET scans using the two radiotracers showed similar kidney, liver, and lung kinetics. A dysregulated tryptophan metabolism in NF1 mice was further confirmed using immunohistostaining. L-[18F]FETrp is warranted to further investigate differentiating malignant NF1 tumors from benign PNFs. The study may reveal the tryptophan-kynurenine pathway as a therapeutic target for treating NF1.

DeepPPAPredMut: deep ensemble method for predicting the binding affinity change in protein-protein complexes upon mutation.

MOTIVATION: Protein-protein interactions underpin many cellular processes and their disruption due to mutations can lead to diseases. With the evolution of protein structure prediction methods like AlphaFold2 and the availability of extensive experimental affinity data, there is a pressing need for updated computational tools that can efficiently predict changes in binding affinity caused by mutations in protein-protein complexes. RESULTS: We developed a deep ensemble model that leverages protein sequences, predicted structure-based features, and protein functional classes to accurately predict the change in binding affinity due to mutations. The model achieved a correlation of 0.97 and a mean absolute error (MAE) of 0.35 kcal/mol on the training dataset, and maintained robust performance on the test set with a correlation of 0.72 and a MAE of 0.83 kcal/mol. Further validation using Leave-One-Out Complex (LOOC) cross-validation exhibited a correlation of 0.83 and a MAE of 0.51 kcal/mol, indicating consistent performance. AVAILABILITY AND IMPLEMENTATION: https://web.iitm.ac.in/bioinfo2/DeepPPAPredMut/index.html.

Clinical application of magnetic resonance elastography in pediatric neurological disorders.

Magnetic resonance elastography is a relatively new, rapidly evolving quantitative magnetic resonance imaging technique which can be used for mapping the viscoelastic mechanical properties of soft tissues. MR elastography measurements are akin to manual palpation but with the advantages of both being quantitative and being useful for regions which are not available for palpation, such as the human brain. MR elastography is noninvasive, well tolerated, and complements standard radiological and histopathological studies by providing in vivo measurements that reflect tissue microstructural integrity. While brain MR elastography studies in adults are becoming frequent, published studies on the utility of MR elastography in children are sparse. In this review, we have summarized the major scientific principles and recent clinical applications of brain MR elastography in diagnostic neuroscience and discuss avenues for impact in assessing the pediatric brain.

Deep learning-based method for predicting and classifying the binding affinity of protein-protein complexes.

Protein-protein interactions (PPIs) play a critical role in various biological processes. Accurately estimating the binding affinity of PPIs is essential for understanding the underlying molecular recognition mechanisms. In this study, we employed a deep learning approach to predict the binding affinity (ΔG) of protein-protein complexes. To this end, we compiled a dataset of 903 protein-protein complexes, each with its corresponding experimental binding affinity, which belong to six functional classes. We extracted 8 to 20 non-redundant features from the sequence information as well as the predicted three-dimensional structures using feature selection methods for each protein functional class. Our method showed an overall mean absolute error of 1.05 kcal/mol and a correlation of 0.79 between experimental and predicted ΔG values. Additionally, we evaluated our model for discriminating high and low affinity protein-protein complexes and it achieved an accuracy of 87% with an F1 score of 0.86 using 10-fold cross-validation on the selected features. Our approach presents an efficient tool for studying PPIs and provides crucial insights into the underlying mechanisms of the molecular recognition process. The web server can be freely accessed at https://web.iitm.ac.in/bioinfo2/DeepPPAPred/index.html.

Abusive Head Trauma Animal Models: Focus on Biomarkers.

Abusive head trauma (AHT) is a serious traumatic brain injury and the leading cause of death in children younger than 2 years. The development of experimental animal models to simulate clinical AHT cases is challenging. Several animal models have been designed to mimic the pathophysiological and behavioral changes in pediatric AHT, ranging from lissencephalic rodents to gyrencephalic piglets, lambs, and non-human primates. These models can provide helpful information for AHT, but many studies utilizing them lack consistent and rigorous characterization of brain changes and have low reproducibility of the inflicted trauma. Clinical translatability of animal models is also limited due to significant structural differences between developing infant human brains and the brains of animals, and an insufficient ability to mimic the effects of long-term degenerative diseases and to model how secondary injuries impact the development of the brain in children. Nevertheless, animal models can provide clues on biochemical effectors that mediate secondary brain injury after AHT including neuroinflammation, excitotoxicity, reactive oxygen toxicity, axonal damage, and neuronal death. They also allow for investigation of the interdependency of injured neurons and analysis of the cell types involved in neuronal degeneration and malfunction. This review first focuses on the clinical challenges in diagnosing AHT and describes various biomarkers in clinical AHT cases. Then typical preclinical biomarkers such as microglia and astrocytes, reactive oxygen species, and activated N-methyl-D-aspartate receptors in AHT are described, and the value and limitations of animal models in preclinical drug discovery for AHT are discussed.

DeepBSRPred: deep learning-based binding site residue prediction for proteins.

MOTIVATION: Proteins-protein interactions (PPIs) are important to govern several cellular activities. Amino acid residues, which are located at the interface are known as the binding sites and the information about binding sites helps to understand the binding affinities and functions of protein-protein complexes. RESULTS: We have developed a deep neural network-based method, DeepBSRPred, for predicting the binding sites using protein sequence information and predicted structures from AlphaFold2. Specific sequence and structure-based features include position-specific scoring matrix (PSSM), solvent accessible surface area, conservation score and amino acid properties, and residue depth, respectively. Our method predicted the binding sites with an average F1 score of 0.73 in a dataset of 1236 proteins. Further, we compared the performance with other existing methods in the literature using four benchmark datasets and our method outperformed those methods. AVAILABILITY AND IMPLEMENTATION: The DeepBSRPred web server can be found at https://web.iitm.ac.in/bioinfo2/deepbsrpred/index.html , along with all datasets used in this study. The trained models, the DeepBSRPred standalone source code, and the feature computation pipeline are freely available at https://web.iitm.ac.in/bioinfo2/deepbsrpred/download.html .

Epilepsy and Electroencephalogram Characteristics in Children with Neurofibromatosis Type 1, What We Have Learned from a Tertiary Center Five Years' Experience.

Introduction: Neurofibromatosis type 1(NF-1) is the commonest neurocutaneous phacomatosis in children. Epilepsy is an infrequent comorbidity. Reports of seizure and Electroencephalogram (EEG) characteristics in children are sparse. Methods: A retrospective review was performed on patients with NF-1 seen between 2016-2020. Patients with co-existing epilepsy were identified. Demographic, clinical, radiological and neurophysiological data were reviewed and analyzed. Results: Out of 118 children with NF1, 16 had epilepsy. 11 patients had focal onset seizures, whereas 5 had generalized onset seizures. Most patients had easy seizure control. Focal epileptiform discharges were the most prevalent EEG abnormality. There was no significant correlation between seizure patterns and presence of intracranial tumors. Conclusion: Epilepsy is a relatively uncommon in pediatric NF-1. Seizures are often of focal semiology and likely to be easily controlled. Focal and multifocal spike epileptiform discharges are the typical interictal EEG findings. Correlation of clinical and EEG findings with intracranial lesions is poor.

Aortopulmonary artery fistula: A rare complication of balloon dilatation of the pulmonary artery.

Acquired aortopulmonary fistula (APF) in the setting of repaired congenital heart disease is extremely rare but potentially fatal, so timely diagnosis and treatment are critical. We present a case of an 8-year-old female with a history of complex Taussig-Bing anomaly, who underwent an arterial switch procedure with LeCompte maneuver and ventricular septal defect closure early in life. The patient developed neopulmonary stenosis and branch pulmonary artery (PA) stenosis, for which she underwent patch augmentation and balloon dilatation of the left PA. The patient presented with a fistula between the ascending aorta and the left branch PA, confirmed by echocardiography and cardiac catheterization. She underwent repair of the APF with a homograft patch reconstruction of the ascending aorta.

Advanced Neuroimaging Approaches to Pediatric Brain Tumors.

Central nervous system tumors are the most common pediatric solid tumors; they are also the most lethal. Unlike adults, childhood brain tumors are mostly primary in origin and differ in type, location and molecular signature. Tumor characteristics (incidence, location, and type) vary with age. Children present with a variety of symptoms, making early accurate diagnosis challenging. Neuroimaging is key in the initial diagnosis and monitoring of pediatric brain tumors. Conventional anatomic imaging approaches (computed tomography (CT) and magnetic resonance imaging (MRI)) are useful for tumor detection but have limited utility differentiating tumor types and grades. Advanced MRI techniques (diffusion-weighed imaging, diffusion tensor imaging, functional MRI, arterial spin labeling perfusion imaging, MR spectroscopy, and MR elastography) provide additional and improved structural and functional information. Combined with positron emission tomography (PET) and single-photon emission CT (SPECT), advanced techniques provide functional information on tumor metabolism and physiology through the use of radiotracer probes. Radiomics and radiogenomics offer promising insight into the prediction of tumor subtype, post-treatment response to treatment, and prognostication. In this paper, a brief review of pediatric brain cancers, by type, is provided with a comprehensive description of advanced imaging techniques including clinical applications that are currently utilized for the assessment and evaluation of pediatric brain tumors.

Utilization of neonatal sedation and anesthesia: an SPR survey.

BACKGROUND: There is little data regarding the use of sedation and anesthesia for neonatal imaging, with practice patterns varying widely across institutions. OBJECTIVE: To understand the current utilization of sedation and anesthesia for neonatal imaging, and review the current literature and recommendations. MATERIALS AND METHODS: One thousand, two hundred twenty-six questionnaire invitations were emailed to North American physician members of the Society for Pediatric Radiology using the Survey Monkey platform. Descriptive statistical analysis of the responses was performed. RESULTS: The final results represented 59 institutions from 26 U.S. states, the District of Columbia and three Canadian provinces. Discrepant responses from institutions with multiple respondents (13 out of 59 institutions) were prevalent in multiple categories. Of the 80 total respondents, slightly more than half (56%) were associated with children's hospitals and 44% with the pediatric division of an adult radiology department. Most radiologists (70%) were cognizant of the neonatal sedation policies in their departments. A majority (89%) acknowledged awareness of neurotoxicity concerns in the literature and agreed with the validity of these concerns. In neonates undergoing magnetic resonance imaging (MRI), 46% of respondents reported attempting feed and bundle in all patients and an additional 46% attempt on a case-by-case basis, with most (35%) using a single swaddling attempt before sedation. Sedation was most often used for neonatal interventional procedures (93%) followed by MR (85%), nuclear medicine (48%) and computed tomography (31%). More than half of respondents (63%) reported an average success rate of greater than 50% when using neonatal sedation for MR. CONCLUSION: Current practice patterns, policies and understanding of the use of sedation and anesthesia for neonatal imaging vary widely across institutions in North America, and even among radiologists from the same institution. Our survey highlights the need for improved awareness, education, and standardization at both the institutional level and the societal level. Awareness of the potential for anesthetic neurotoxicity and success of non-pharmacologic approaches to neonatal imaging is crucial, along with education of health care personnel, systematic approaches to quality control and improvement, and integration of evidence-based protocols into clinical practice.

Para and Post-COVID-19 CNS Acute Demyelinating Disorders in Children: A Case Series on Expanding the Spectrum of Clinical and Radiological Characteristics.

Viral infections can serve as a trigger for variable autoimmune, antibody-mediated demyelinating disorders. There is accumulating evidence that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, causing coronavirus disease 2019 (COVID-19) infection and responsible for the current worldwide pandemic, can lead to a cascade of immune-mediated brain and spinal cord demyelinating injuries. However, such observation in the pediatric age group was only reported in very few patients. Thus, the heterogeneous spectrum of this phenomenon in children is still unfolding. We are reporting a case series of five pediatric patients with a variety of acute central nervous system (CNS) demyelinating disorders in the context of acute or recent COVID-19 infection. A 16-year-old female with anti-myelin oligodendrocyte glycoprotein (MOG) disorder, an eight-year-old male with acute disseminated encephalomyelitis (ADEM), a 13-year-old female with neuromyelitis optica spectrum disorder (NMOSD), and two 14 and 13-year-old females with new-onset multiple sclerosis (MS) are reported, all of whom presented acutely following COVID-19 infection. We propose that para and post-infectious CNS demyelinating disorders can potentially follow acute COVID-19 infection in children. Considering SARS-CoV-2 testing as a part of diagnostic workup is possibly useful. Awareness of the presence of this phenomenon can help in the recognition and management of those patients.

Pearls and Pitfalls in Arterial Spin Labeling Perfusion-Weighted Imaging in Clinical Pediatric Imaging.

Characteristic arterial spin labeling (ASL) perfusion patterns are seen in a wide variety of pediatric brain pathologies, highlighting the potential added value and prognostic role of this magnetic resonance imaging (MRI) perfusion-weighted imaging modality. Our objective is to review the basic clinical physics, technical underpinnings, and artifacts and challenges as we highlight some of the most clinically relevant pathologies to the application of ASL in the pediatric setting.

Illustrative Tutorials for ProThermDB: Thermodynamic Database for Proteins and Mutants.

ProThermDB (https://web.iitm.ac.in/bioinfo2/prothermdb/index.html) is a primary resource for protein stability, which contains experimentally determined thermodynamic data for proteins and their mutants. The most recent version of ProThermDB accumulates the data obtained from both high- and low-throughput experimental biophysical methods. It includes comprehensive information at four different levels, i.e.: (i) protein sequence and structure; (ii) experimental conditions; (iii) thermodynamic parameters such as Gibbs free energy, melting temperature, enthalpy, etc.; and (iv) literature. In the following protocols, we present detailed tutorials for retrieving data using different search, display and sorting options, interpretation of search results, description of each entry-level information category, data upload and download, cross-links with other databases, and visualization options. This protocol consists of six pictorial exercises, which are useful for biologists/users to understand the contents and organization of data in ProThermDB. Further, potential applications of ProThermDB in protein engineering are discussed. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Retrieval of experimental thermodynamic data for wild-type and mutants of a specific protein using a simple query Basic Protocol 2: Retrieval of stabilizing point mutations, which are located at the interior of α-helical regions, and obtaining data by thermal denaturation methods Basic Protocol 3: Retrieval of destabilizing point mutations, which are in ß-sheets of exposed regions, and obtaining data by chemical denaturation methods (urea and GdnHCl) Basic Protocol 4: Retrieval of stabilizing and destabilizing point mutations in a range of physiological conditions (pH: 6-9 and T: 20°C-25°C) and publication years (2010-2020) Support Protocol: Downloading the entire data of the database for academic research purposes and submission of new data in ProThermDB.

Radiosynthesis of 1-(2-[18F]Fluoroethyl)-L-Tryptophan using a One-pot, Two-step Protocol.

The kynurenine pathway (KP) is a primary route for tryptophan metabolism. Evidence strongly suggests that metabolites of the KP play a vital role in tumor proliferation, epilepsy, neurodegenerative diseases, and psychiatric illnesses due to their immune-modulatory, neuro-modulatory, and neurotoxic effects. The most extensively used positron emission tomography (PET) agent for mapping tryptophan metabolism, α-[11C]methyl-L-tryptophan ([11C]AMT), has a short half-life of 20 min with laborious radiosynthesis procedures. An onsite cyclotron is required to radiosynthesize [11C]AMT. Only a limited number of centers produce [11C]AMT for preclinical studies and clinical investigations. Hence, the development of an alternative imaging agent that has a longer half-life, favorable in vivo kinetics, and is easy to automate is urgently needed. The utility and value of 1-(2-[18F]fluoroethyl)-L-tryptophan, a fluorine-18-labeled tryptophan analog, has been reported in preclinical applications in cell line-derived xenografts, patient-derived xenografts, and transgenic tumor models. This paper presents a protocol for the radiosynthesis of 1-(2-[18F]fluoroethyl)-L-tryptophan using a one-pot, two-step strategy. Using this protocol, the radiotracer can be produced in a 20 ± 5% (decay corrected at the end of synthesis, n > 20) radiochemical yield, with both radiochemical purity and enantiomeric excess of over 95%. The protocol features a small precursor amount with no more than 0.5 mL of reaction solvent in each step, low loading of potentially toxic 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane (K222), and an environmentally benign and injectable mobile phase for purification. The protocol can be easily configured to produce 1-(2-[18F]fluoroethyl)-L-tryptophan for clinical investigation in a commercially available module.

Unravelling neuroinflammation in abusive head trauma with radiotracer imaging.

Abusive head trauma (AHT) is a leading cause of mortality and morbidity in child abuse, with a mortality rate of approximately 25%. In survivors, the prognosis remains dismal, with high prevalence of cerebral palsy, epilepsy and neuropsychiatric disorders. Early and accurate diagnosis of AHT is challenging, both clinically and radiologically, with up to one-third of cases missed on initial examination. Moreover, most of the management in AHT is supportive, reflective of the lack of clear understanding of specific pathogenic mechanisms underlying secondary insult, with approaches targeted toward decreasing intracranial hypertension and reducing cerebral metabolism, cell death and excitotoxicity. Multiple studies have elucidated the role of pro- and anti-inflammatory cytokines and chemokines with upregulation/recruitment of microglia/macrophages, oligodendrocytes and astrocytes in severe traumatic brain injury (TBI). In addition, recent studies in animal models of AHT have demonstrated significant upregulation of microglia, with a potential role of inflammatory cascade contributing to secondary insult. Despite the histological and biochemical evidence, there is a significant dearth of specific imaging approaches to identify this neuroinflammation in AHT. The primary motivation for development of such imaging approaches stems from the need to therapeutically target neuroinflammation and establish its utility in monitoring and prognostication. In the present paper, we discuss the available data suggesting the potential role of neuroinflammation in AHT and role of radiotracer imaging in aiding diagnosis and patient management.

Artificial intelligence in child abuse imaging.

There have been rapid advances in artificial intelligence (AI) technology in recent years, and the field of diagnostic imaging is no exception. Just as digital technology revolutionized how radiology is practiced, so these new technologies also appear poised to bring sweeping change. As AI tools make the transition from the theoretical to the everyday, important decisions need to be made about how they will be applied and what their role will be in the practice of radiology. Pediatric radiology presents distinct challenges and opportunities for the application of these tools, and in this article we discuss some of these, specifically as they relate to the prediction, identification and investigation of child abuse.

Birth-related subdural hemorrhage: prevalence and imaging morphology.

BACKGROUND: Birth trauma accounts for 1-2% of the mortality in newborns with significant intracranial injuries presenting in the immediate postnatal period. However, a significant number of asymptomatic neonates harbor birth-related intracranial hemorrhage (ICH), with birth-related subdural hemorrhage (SDH) being a common occurrence on infant brain CT and MRI studies performed as a standard of care for a variety of reasons. Although clinically insignificant, birth-related SDH is frequently brought up in courts as an alternative explanation for SDH in suspected abusive head trauma. OBJECTIVE: The aim of this study was to determine prevalence, imaging morphology and distribution of birth-related SDHs on brain CT and MRI studies obtained as a standard of care in infants up to 1 month old. We further tried to ascertain the relationship of birth-related SDHs with mode of delivery and birth weight. MATERIALS AND METHODS: Infants up to the age of 1 month who had CT or MRI of the brain performed between Jan. 1, 2018, and March 29, 2020, were included in this retrospective observational study. In addition to the imaging data, we reviewed clinical history, birth history including birth weight and mode of delivery, and final diagnoses. RESULTS: Two hundred six infants younger than 30 days (range 0-29 days, mean 11.9 days, median 11 days and standard deviation [SD] 8.4 days) had a CT or MRI study during the study period. Among these, 58 infants were excluded as per the exclusion criteria. Among the included 148 infants, 88 (59.5%) had no imaging evidence of SDH. An additional 56 (37.8%) infants were assessed as having birth-related SDH based on review of clinical data. Within the birth-related SDH cohort (56 infants), only supratentorial SDH was identified in 5 (8.9%), only infratentorial SDH was identified in 14 (25%), while SDHs within both compartments were identified in 37 (66.1%) infants. The most common location for supratentorial birth-related SDH was along the occipital lobes (31/42, 73.8%), with other common locations being along the posterior interhemispheric fissure (30/42, 71.4%) and fronto-parietal convexity (9/42, 21.4%). The distribution of posterior fossa SDH was along the tentorium (38/51, 74.5%), along the cerebellum (38/51, 74.5%) and in both the locations (25/51, 49.0%). The rate of SDH was significantly higher in vaginal delivery group (46/84, 54.7%) as compared to caesarean section group (10/57, 17.5%) (P<0.05). We did not find any statistically significant difference between the birth weights of normal and birth-related SDH cohorts (P>0.05). CONCLUSION: Birth-related SDH is a common occurrence, with our study suggesting a prevalence of 37.8%. The most common distribution of birth-related SDH is within both the supra- and infratentorial compartments (66.1%) followed by infratentorial compartment (25%). The rate of birth-related SDH was significantly higher in vaginal delivery group as compared to caesarean section group.

Folate Receptor-Mediated siRNA Delivery: Recent Developments and Future Directions for RNAi Therapeutics.

RNA interference (RNAi), a gene regulatory process mediated by small interfering RNAs (siRNAs), has made remarkable progress as a potential therapeutic agent against various diseases. However, RNAi is associated with fundamental challenges such as poor systemic delivery and susceptibility to the nucleases. Targeting ligand-bound delivery vehicles has improved the accumulation of drug at the target site, which has resulted in high transfection efficiency and enhanced gene silencing. Recently, folate receptor (FR)-mediated targeted delivery of siRNAs has garnered attention due to their enhanced cellular uptake and high transfection efficiency toward tumor cells. Folic acid (FA), due to its small size, low immunogenicity, high in vivo stability, and high binding affinity toward FRs, has attracted much attention for targeted siRNA delivery. FRs are overexpressed in a large number of tumors, including ovarian, breast, kidney, and lung cancer cells. In this review, we discuss recent advances in FA-mediated siRNA delivery to treat cancers and inflammatory diseases. This review summarizes various FA-conjugated nanoparticle systems reported so far in the literature, including liposome, silica, metal, graphene, dendrimers, chitosan, organic copolymers, and RNA nanoparticles. This review will help in the design and development of potential delivery vehicles for siRNA drug targeting to tumor cells using an FR-mediated approach.

Idiopathic midaortic syndrome.

Midaortic syndrome (MAS) is characterized by narrowing of the descending aorta between the distal aortic arch and the aortic bifurcation. We present the case of a 4-year-old male presenting with a murmur and diagnosed with MAS. He was treated with a thoracoabdominal bypass graft.

ProThermDB: thermodynamic database for proteins and mutants revisited after 15 years.

ProThermDB is an updated version of the thermodynamic database for proteins and mutants (ProTherm), which has ∼31 500 data on protein stability, an increase of 84% from the previous version. It contains several thermodynamic parameters such as melting temperature, free energy obtained with thermal and denaturant denaturation, enthalpy change and heat capacity change along with experimental methods and conditions, sequence, structure and literature information. Besides, the current version of the database includes about 120 000 thermodynamic data obtained for different organisms and cell lines, which are determined by recent high throughput proteomics techniques using whole-cell approaches. In addition, we provided a graphical interface for visualization of mutations at sequence and structure levels. ProThermDB is cross-linked with other relevant databases, PDB, UniProt, PubMed etc. It is freely available at https://web.iitm.ac.in/bioinfo2/prothermdb/index.html without any login requirements. It is implemented in Python, HTML and JavaScript, and supports the latest versions of major browsers, such as Firefox, Chrome and Safari.