Package installation is easy for experienced users, but novices sometimes struggle with dependencies and compilers. This presents a barrier that can hinder the otherwise broad adoption of new tools. We present Scoria, a Python package for manipulating three-dimensional molecular data. Unlike similar packages, Scoria requires no dependencies, compilation, or system-wide installation. One can incorporate the Scoria source code directly into their own programs. But Scoria is not designed to compete with other similar packages. Rather, it complements them. Our package leverages others , if present, to speed and extend its own functionality. To show its utility, we use Scoria to analyze a molecular dynamics trajectory. Our FootPrint script colors the atoms of one chain by the frequency of their contacts with a second chain. We are hopeful that Scoria will be a useful tool for the computational-biology community. A copy is available for download free of charge (Apache License 2.0) at The online version of this article (doi:10.1186/s13321-017-0237-8) contains supplementary material, which is available to authorized users. Even small usability barriers hinder the widespread adoption of new computational tools. When an experienced computational biologist wishes to analyze 3D molecular data, she might turn to popular Python packages such as MDAnalysis . These pPython, a popular and powerful programming language for scientific computing, is an interpreted language with easy-to-read, intuitive syntax. The real power of Python comes from its extendibility. Rich Python-package ecosystems  extend its functionality. New Python classes and functions expose libraries written in faster languages such as C. Unfortunately, this extendibility can impact usability. Many computationalists use packages that depend on NumPy and SciPy, add-ons that speed mathematical operations. These in turn often require the installation of a C compiler. NumPy is incompatible with PyPy, a popular just-in-time Python-2.7 compiler that is faster than the standard Python executable. If a program uses a NumPy-dependent package only lightly, any speed NumPy provides may be offset by the slowness of standard Python. And third-party packages are not universally supported on all versions of Python and on all operating systems.These challenges limit many existing molecular-modeling packages. The excellent package MDAnalysis is a good example. It depends on NumPy and so is incompatible with PyPy, it does not support the Windows operating system, and it is only partially compatible with Python 3 . Packages like MDAnalysis are also complex because of their many powerful features. While well suited for many projects, some projects need only more basic operations.scoria directory into their own Python project to use it. (3) It is compatible with Python 2, Python 3, and PyPy. (4) It runs on all major operating systems .We have developed a set of functions over the last several years in service of our own published software projects –14. We hrequired dependencies, Scoria does use NumPy and SciPy installations, if present, to speed and extend its own functionality. A separate, derivative version of Scoria, called Scoria MDA, can additionally leverage MDAnalysis. Scoria thus provides the best of both worlds. It is a simple, independent package for those who need basic functionality, but it provides compatibility with external libraries for those who need speed.Though it has no Scoria is useful for both analyzing molecular dynamics (MD) trajectories and molecular modeling. For example, we have used beta-version Scoria functions to create large-scale lipid-bilayer models , to consscoria directory directly into their projects; alternatively, they can install Scoria via popular package managers . Docstrings describe the key functions, show proper usage, and present informative tables. The package itself, as well as an HTML help file generated from the docstrings, can be found online at http://durrantlab.com/scoria/.We hope that Scoria will be a useful open-source tool for the community. Users can copy the Scoria provides functions that read and write several file types. It natively supports PDBQT as well as single and multi-frame PDB formats. Scoria also has its own single-frame molecular file format, the PYM format. PYM files have optimized read/write speeds for use in high-throughput computational projects. Finally, Scoria MDA can import other file types via MDAnalysis, both as file lists and from MDAnalysis Universe objects.Molecule objects. Scoria includes functions for calculating distances, steric clashes, and root-mean-square deviations between molecules.Scoria stores molecular and atomic information. It tracks individual atom names, residues, chain ids, residue sequences, occupancies, temperature factors, elements, charges, atomic coordinates, and trajectories. The user can perform calculations on the entire molecule or on subsets of atoms. Selections by atomic properties, molecular affiliations, and spatial properties define these subsets. The user can also specify custom selection criteria based on any stored atom, residue, or chain data. The user can translate and rotate molecular coordinates. Rotations about single points and lines are possible. Aside from using 3D coordinates to define these points and lines, the user can also specify atoms. In the latter case, Scoria uses the atomic coordinates. The user can also compare, contrast, align, and merge two Molecule class. After creating a molecule object , the user accesses the functions via the mol.fileio, mol.information, mol.selections, mol.manipulation, and mol.other_molecule namespaces. For convenience, users can also access these functions directly from the Molecule object, e.g., mol.save_pdb is identical to mol.fileio.save_pdb.For ease of use, the above functions are all accessible from the main To show how Scoria can be used to analyze a molecular dynamics (MD) simulation, we ran a simulation of the rock1-dimer/shroom2 complex using Amber , 17 on ttleap program [ff14SB [ionsjc_tip3p force fields [The AmberTools  program  paramete [ff14SB  and ionse fields . We miniWe then restrained most of the protein backbone and heated the system. We first heated from 0 to 100 K at constant volume, and then from 100 to 300 K at constant pressure. The simulation continued at 300 K, constant pressure, during a third step of the heating phase. Following heating, the system was further equilibrated with the same backbone restraints. We then removed all restraints for a second round of equilibration. 50 ns of productive simulation followed, which were used for analysis.Scoria is a Python package that can load, save, analyze, and manipulate 3D molecular models without requiring compilation, dependencies, or system-wide installation. Scoria’s use of NumPy and SciPy is optional; most of the basic features of the package do not require any dependencies. Extra features are available if NumPy or SciPy are present. A separate, derivative version (Scoria MDA) similarly integrates MDAnalysis support.We have tested Scoria’s basic functionality on all major operating systems using both Python and PyPy. Table Scoria’s basic features are written in pure Python. But when the user needs more intensive calculations , she can install NumPy and SciPy. When she wishes to use binary formats , she can use Scoria’s MDAnalysis-enabled version. Table File input/output is often the greatest bottleneck when analyzing large numbers of molecular models. Scoria supports reading and writing to several formats, so we performed several benchmarks. As a test PDB file, we used 51 consecutive residues (401 atoms) taken from the 1XDN structure . To calcScoria saved to a binary PYM file 2.3 times faster than to the equivalent text-formatted PDB file, and it loaded the PYM file 12.7 times faster than the PDB. PYM files are thus well suited to high-throughput projects when fast file I/O is critical.The Python executable and installed dependencies also affected load and save times. We tested two different executables: the standard Python interpreter  and PyPy, which uses just-in-time compilation to speed run times. We also tested CPython installations with and without dependencies such as NumPy. Loading and saving to the PYM and MDAnalysis formats requires NumPy, so only the PDB format is useful for comparing these three Python setups.When saving the PDB file, PyPy was the fastest, despite lacking dependencies such as NumPy and SciPy. PyPy saved the test file 1.5 times faster than CPython with dependencies installed, which was in turn 1.5 times faster than CPython when those dependencies were absent. When loading the test PDB file, dependencies such as NumPy allowed CPython to load 6.2 faster than CPython without dependencies installed. PyPy support for NumPy is incomplete, but even so CPython/NumPy was only 1.9 times faster than PyPy (without NumPy).It is easy to imagine scenarios in which PyPy would be the fastest choice. For example, we loaded the test file without calculating bonds by distance and saved 500 copies to disk . PyPy finished 3.1 times faster than CPython with NumPy (2.2 vs. 7.1 s) because this test script greatly favors output  over input (loading).PyPy is also faster when code includes time-consuming features that are NumPy independent. To illustrate, we wrote a script that performed 50 million variable assignments before using Scoria to load the test PDB file. CPython/NumPy loaded the molecular model faster, but PyPy accelerated variable assignment and so ran 8.3 times quicker overall (0.3 vs. 2.8 s).To show Scoria’s utility, we created a simple script that calculates molecular “footprints.” When simulating multi-domain systems, users may wish to identify inter-domain contacts. Surfaces with high contact residence times are likely to participate in critical inter-domain interactions. These regions may be good targets for mutagenesis studies aiming at disrupting protein–protein interfaces.The script first loads a simulated trajectory and divides it into two parts . For each atom, it calculates a contact residence time by considering how often that atom comes within 3 Å of the other domain. Scoria stores these residence times in the occupancy fields of each atom and saves the molecular data to a PDB file. VMD  is then We applied this script to a brief MD simulation of the rock1-dimer/shroom2 complex. Previous work has shown that shroom proteins bind rho kinase (rock), regulating rock distribution and activity , 24, 25.Figure 1834SLSGRLA1840 and 1878LKENLDRR1885. These two regions are highly homologous to two human shroom2 regions , yielding 80% accuracy overall.The shroom2 mutations L1501A, L1548A, and K1487A are also critical . Our anaWhile imperfect, this contact-footprint method may be useful in prospective studies aimed at prioritizing residues for mutagenesis. Regardless, that such a tool could be constructed in a few lines of code demonstrates Scoria’s utility.Scoria is a Python package for simple molecular modeling and data-collection tasks that need a light overhead. It can improve the usability of new analysis tools, encouraging broader adoption among end users who are uncomfortable installing Python dependencies.Unlike some similar packages, Scoria is compatible with the Windows operating system as well as PyPy. If a script performs only limited molecular manipulation, the speed benefits of PyPy make Scoria an attractive option.We will continue to add new functionality to Scoria per our ongoing projects’ needs. Additions will be broadly useful; simple (to prevent codebase bloating); and, to the extent possible, free of any required dependencies. As Scoria is an open-source project, we will make future changes publically available. Contributions from other users that meet these same inclusion criteria are welcome.We have used Scoria components in other published software packages –14 underAdditional file 1: Table S1. Main Scoria functions, with associated dependencies (if any).Additional file 2. An archived version of Scoria, without MDAnalysis support.Additional file 3. An archived version of Scoria, derived from the main Scoria branch, that includes MDAnalysis support.


In 1974, molecular electronics pioneers Mark Ratner and Arieh Aviram predicted that a single molecule could act as a diode, in which electronic current can be rectified. The electronic rectification property of the diode is one of basic functions of electronic components and since then, the molecular diode has been investigated as a first single-molecule device that would have a practical application. In this review, we first describe the experimental fabrication and electronic characterization techniques of molecular diodes consisting of a small number of molecules or a single molecule. Then, two main mechanisms of the rectification property of the molecular diode are discussed. Finally, representative results for the molecular diode are reviewed and a brief outlook on crucial issues that need to be addressed in future research is discussed. The diode is one of the basic components of electric devices. The diode acts as a rectifier, permitting electronic current to flow only in one direction. Rectification of electronic current was first achieved in a vacuum diode, then later in junctions where a p-type semiconductor is contacted with an n-type semiconductor . RectifiThe molecular diode was first prepared by Langmuir-Blodgett (LB) technique where molecularly thin LB films were sandwiched by top and bottom metal electrodes  and the I-V measurement of the single-molecule junction is performed by positioning a AFM tip atop the Au nanoparticle . A. A44]. AI–V response of single native DNA and DNA-coralyne complex, where the tip was grounded, and the bias voltage was applied to the substrate. The native DNA single molecular junction did not show rectification behavior, while the DNA-coralyne single molecular junction showed rectification with a high rectification ratio around 15 at 1.1 V. This rectification ratio was close to the theoretically estimated upper limit of 20 that could be achieved in a coherent transport molecular junction system. The theoretical study revealed that the rectification behavior was caused by the coralyne induced local spatial asymmetry of the distribution of electron states along the DNA chain. The wavefunction weights at the left edge of the molecule were 10,000 times larger than that at the right edge. This asymmetry of the distribution of electron states led to a change in the coupling between the HOMO-1 orbital and the electrode when an external voltage was applied.Along with the studies on the single-molecule diodes of the small organic molecules, single-molecule diodes based on biological large molecules such as DNA have been investigated owing to the inherent structural and molecular recognition properties that could be ideal for molecular electronics applications. Guo et al. fabricated a DNA-based rectifier with high rectification property using STM-BJ  Figure a. The DN2 and triphenylene 3 pair or a dimer of 3, in which the enclosed aromatic pair is bookended by the electron-poor triazine panels 4 of the cage. The empty cage 1, homo π-stacked complex 1·(3·3), and hetero π-stacked complex 1·(2·3) behave like an insulator, a resistor, and a rectifier, respectively. STM-BJ measurements revealed that both the homo and hetero π-stacked complexes 1·(3·3) of 1·(2·3) indicated conductive characteristics with electronic conductance values of 10−3–10−2 G0, while the empty cage exhibited an insulating characteristic with ~10−5 G0. STM-BJ-based I–V measurements were performed to confirm the electronic functionality of the π-stacked complexes, for which an additional diode property with a rectification ratio of 1.4–2.0 was determined for the hetero π-stacked complex 1·(2·3), as shown in 1·(2·3) and 1·(3·2)) against the electron-transport directions and the corresponding lowest unoccupied molecular orbital conduction channels localized on one side of the molecule 2 in the molecular junction.The single-molecule diode with a host–guest system has been demonstrated using a self-assembled molecular cage containing aromatic stacks . In typiIn this review, we described the technique that measures the electron transport properties of molecular junctions with a small number of molecules and a single molecule. On the basis of LB and self-assembly methods in combination with the liquid metal electrode, the molecular orientation controlled diode with a small number of molecules can be fabricated. The MCBJ and STM-BJ technique enables us to prepare the single-molecule junction. In this review, we focused on the rectification properties of the molecular diode. The rectification properties were initially proposed for the molecular junction, where the donor and acceptor units were connected via an insulating unit (Aviram-Ratner (A-R) model). Recent experimental studies revealed that the rectification properties appeared when the conduction orbital was strongly hybridized with one of the metal electrodes, in addition to the A-R type molecular diode. High rectification ratio of more than 100 was reported for the molecular diode based on the asymmetric metal-molecule coupling. Recent developments revealed that the structural degree of the freedom of the molecule(s), the surface morphology of the metal electrodes, and environment of the molecular junctions crucially affect the rectification properties of the molecularly thick junctions and the single-molecule junctions. The measurement technique of a small number of molecules has been improved in recent years. Currently, thermopower, force, atomic and electronic structures in the molecular junctions can be studied with advanced measurement techniques on the single molecule scale and link themselves to the performance and electronic function of devices. Precise control over the chemistry of the molecular backbone as well as the metal-molecular interface structures are crucial for understanding the unique electronic function of the molecular junctions, which could be an important aspect of fabricating future single-molecule-based electronic devices.


This review summarizes current concepts in the diagnosis and management of the patients with eosinophilic granuloma. Given the benign biology, the clinical course, and the pediatric group of patients that this condition more commonly affects, a treatment approach that carries a lower risk of complications while ensuring a successful cure is desirable. Variable treatment options have been reported with satisfactory results and a recurrence rate of less than 20 %. In this setting, symptomatic lesions that are accessible in the spine or the extremities may be treated with intralesional methylprednisolone injection after tissue biopsy for histological diagnosis. Langerhans-cell histiocytosis is a rare disease involving clonal proliferation of Langerhans cells , 2. It iEosinophilic granuloma is a rare, benign tumor-like disorder characterized by clonal proliferation of antigen-presenting mononuclear cells of dendritic origin known as Langerhans cells , 2. It iThe typical radiographic appearance of eosinophilic granuloma of the extremities is a punched-out lytic-bone lesion without reactive sclerosis. In most cases, a hypervascularized soft-tissue mass surrounds the affected bone , 27. TheTissue biopsy for histological diagnosis is necessary when clinical and radiological manifestations are ambiguous, and the lesions are symptomatic . CT-guidVarious treatment options have been reported for eosinophilic granuloma of bone, including observation and immobilization, indomethacin administration, methylprednisolone injections, radiofrequency ablation, local excision and curettage with or without bone grafting, chemotherapy and irradiation; results have been reported as satisfactory with a recurrence rate of less than 20 % , 43–48. Since eosinophilic granuloma in children is known to resolve spontaneously with time, observation alone or biopsy alone to confirm the diagnosis have also been recommended as a treatment strategy . A previGiven the benign biology and clinical course of eosinophilic granuloma and the pediatric group of patients that this condition more commonly affects, a treatment approach that carries a lower risk of complications while ensuring a successful cure is desirable. In this setting, symptomatic lesions that are accessible in the spine Figs. , 2, 3 orLangerhans histiocytosis, as a systemic disease, appears to be one of the most tissue-destructive syndromes, able to induce multiple and grossly apparent lytic lesions involving many organs and the bones. Since this lytic activity cannot be connected with a neoplastic nature of the disease, one can reasonably assume that histiocytosis X cells induce bone resorption in eosinophilic granuloma through their ability to secrete locally tissue-lytic factors such as interleukins and prostaglandins . SeveralScaglietti et al.  first reComplications such as femoral osteomyelitis  and obstThis review summarizes current concepts in the diagnosis and management of patients with eosinophilic granuloma, with emphasis on the role of intralesional methylprednisolone injection for the successful cure of patients with symptomatic lesions. In the past, we planned for observation alone for patients with imaging evidence of eosinophilic granuloma, and curettage for the most painful lesions. It was our initial belief that percutaneous techniques do not provide adequate tissue for definitive diagnosis for mesenchymal tumors –63. This


The aberrant salience hypothesis of schizophrenia proposes that symptoms such as paranoia arise when behavioural salience is attributed to neutral stimuli. Mesolimbic dopamine dysfunction is thought to be central to this mechanism; building on findings that activity in this pathway conveys a (signed) reward prediction error signal. Given that many psychotic symptoms are not explicitly related to reward learning, it is relevant that recent studies in rodents have demonstrated a role for midbrain dopamine neurons in value-neutral associative learning. Direct evidence for this role in humans, however, is lacking.In this study we asked whether the mesolimbic dopamine circuit is involved in encoding the value-neutral meaningful information of observations, using a model-based functional magnetic resonance imaging (fMRI) task and dopamine positron emission tomography (PET). We define ‘meaningful information’ as the degree to which an observation results in a belief-update to an agent’s internal model of the environment .Participants were tasked to infer the current (hidden) state of the environment, using partially-informative observations at each trial, and then report their belief at the end of each trial. Participant beliefs were modelled using a Hidden Markov Model of the task and iterative application of Bayes’ rule, allowing us to quantify the Bayesian surprise (meaningful information content) associated with a trial observation. Crucially, our task de-correlated Bayesian surprise from both the pure sensory unexpectedness of an observation (unexpected but meaningless information) and its signed reward prediction error. 39 healthy participants  performed 180 task trials within an fMRI scanner. 36 participants also had a [11C]-(+)-4-propyl-9-hydroxy-naphthoxazine (PHNO) PET scan to quantify dopamine-2/3 receptor (D2/3R) availability. 17 participants additionally had a second PET scan 3hrs post 0.5mg/kg oral dexamphetamine, to quantify striatal dopamine release capacity. Neuroimaging analyses were restricted to the bilateral substantia nigra/ventral tegmental area (SN/VTA) and ventral striatum (VS).Our computational model closely predicted participant behaviour (R2= .67), and there was a negative correlation between subclinical paranoia and the degree to which participant behaviour approximated normative Bayesian performance . Neuronal activation encoding the meaningful information content of an observation (Bayesian surprise) was present in SN/VTA and VS (both P(peak)<0.05, SVC), whereas no such encoding was present for sensory unexpectedness or reward-prediction error. Crucially, activation encoding Bayesian surprise was inversely correlated with D2/3R availability in the SN/VTA , consistent with a tonic inhibitory role for midbrain D2/3Rs. Moreover, activation encoding Bayesian surprise was inversely related to dopamine release capacity in the VS , indicating that subjects with high dopamine release capacity showed blunted striatal activation in response to belief-changing information, as is also found in schizophrenia.We provide direct evidence in humans that a mesolimbic dopamine circuit is involved in encoding the meaningful information content of observations, distinct from its involvement in processing signed reward prediction error. These results implicate dopamine in a wider range of function than reward learning, including updating a predictive associative model of the world, and are therefore relevant for the aberrant salience hypothesis of schizophrenia.


The synthetic CH3NH3PbI3 nanofibers were characterized by X-ray diffraction, scanning electron microscopy, thermogravimetric analysis, and photoluminescence. As counter electrodes, the synthesized nanofibers increased the performance of the dye-sensitized solar cells from 1.58 to 2.09%. This improvement was attributed to the enhanced smoothness and efficiency of the electron transport path. Thus, CH3NH3PbI3 perovskites nanofibers are potential alternative to platinum counter electrodes in dye-sensitized solar cells.CH Among these materials, semiconducting CHttention . These nttention –3. Spin-reported –6. Notabreported , 7.Currently, dye-sensitized solar cells (DSSCs) have been extensively studied because of their availability and low cost . In DSSC2, ZnO, and Cu2ZnSnS4 , can be used in solar cells [3NH3PbI3 perovskites are similar to those of CZTSs. Thus, we explore the use of CH3NH3PbI3 perovskite nanofibers as counter electrodes in DSSCs.One-dimensional (1D) nanofibers have attracted considerable attention because of their large length-to-diameter ratio, high surface area, excellent aspect ratio, and effective electronic properties . Throughar cells –14. More3NH3PbI3 perovskite nanofiber using an electrospinning technique. We used polyvinylpyrrolidone (PVP) as an electrospinning medium. Subsequently, to explore the application of the synthesized nanofibers, we attempted to use them as a replacement for Pt in DSSCs.As far as we know, we are the first to fabricate CH3NH3PbI3 precursor was prepared by adding CH3NH3I3 and PbI2 at a ratio of 1:1 to 2 mL N,N-dimethylformamide (DMF) (J&K Scientific Ltd.). Approximately 0.25 g of PVP  was then dissolved into the solution. The resulting solution was stirred for 30 min until a homogeneous mixture was obtained. Facile electrospinning technique method was used to fabricate the PVP-CH3NH3PbI3 perovskite nanofibers. The experimental procedure is schematically illustrated in Fig. 3NH3PbI3 nanofibers obtained were calcined at 150 and 200 °C separately in a nitrogen atmosphere for 5 min.CH2 F20, USA). Thermal analyses were performed through thermogravimetric analysis  in a nitrogenous atmosphere. The band gap was measured through the photoluminescence (PL) spectrum.The phase structures of the prepared samples were characterized using an X-ray diffractometer . Field-effect scanning electron microscope (Hitachi S-520) and transmission electron microscope were then used to study the microstructure of the samples , (220), (310), (224), and (314) crystal planes of the tetragonal perovskite structure. This result is consistent with the previously published results [Figure 3NH3PbI3 perovskite nanofibers at different annealing temperatures. Figure 3NH3PbI3 perovskite nanofibers, which are covered throughout the surface. Moreover, the surfaces of CH3NH3PbI3 nanofibers are quite smooth and their diameters ranges from 140 to 170 nm. Figure 3NH3PbI3 nanofibers. The appearance of multiple diffraction rings is due to the random orientation of the polycrystallites. The spotty ring pattern with missing periodicity is due to the random orientation of the particles [3NH3PbI3 nanofibers after annealing treatment at 150 °C are shown in Fig. 3NH3PbI3 perovskite.Figure articles . The res2 atmosphere. The curve indicates that the thermal decomposition for the as-spun nanofibers is completed in two distinct steps. In the first step, the weight loss (3%) is observed between 25 and 200 °C. The loss is due to the evaporation of water and alcohol. In the second step, (200–400 °C), the weight loss is approximately 35% and is due to PVP degradation, which involves intra- and intermolecular transfer reaction mechanisms.The TGA curves are shown in Fig. 3NH3PbI3 nanofiber at different annealing temperatures is shown in Fig. 3NH3PbI3 layer is comparable to the thickness of the layer [The band gap of the semiconductor plays an essential role in counter electrode of the DSSCs . The PL he layer .Fig. 5PL3NH3PbI3 nanofiber has been applied as a counter electrode in DSSCs. The DSSCs are each equipped with a TiO2/FTO working electrode, redox couple (I−/I3−), and CH3NH3PbI3/FTO counter electrodes [3NH3PbI3/FTO counter electrodes. Figure VOC), short circuit current density (JSC), and filled factor (FF) of the device are 0.45 V, 7.78 mA/cm2, and 45%, respectively. When the counter electrodes have been changed from Pt/FTO to CH3NH3PbI3/FTO, the JSC and FF values have increased slightly by 9.81 mA/cm2 and 51%, respectively. Moreover, the conversion efficiency of the device has increased from 1.58 to 2.09% because of the improved efficiency of the electron transport path in the CH3NH3PbI3/FTO electrodes. Meanwhile, the nanofibers with large surface areas contribute to the redox reaction between the counter electrode and the electrolyte, and thus, a decreased interfacial recombination in the DSSCs is observed [The CHectrodes . The curobserved .Fig. 6J3NH3PbI3 nanofibers with diameters ranging from 140 to 170 nm via electrospinning technique. The XRD analysis results revealed that the synthetic nanofibers contained the pure phase of CH3NH3PbI3 perovskites with good crystallinity. The PL properties demonstrated that the nanofibers have a band gap energy of approximately 1.6 eV. When the nanofibers were used to the counter electrodes of the DSSCs, the conversion efficiency of the device increased from 1.58 to 2.09% because of the large surface area of the small nanofibers. Thus, our synthetic method can significantly contribute to low-cost and large-scale preparation of nanofibers for actual photovoltaic applications.In summary, we successfully synthesized CH


Background: Alzheimer's disease (AD), is a progressive neurodegenerative disease that is characterized by cognitive loss. Most researchers believe that aggregation and accumulation of β-amyloid peptides (Aβ) in brain cells are the central pathological hallmark of this disease.Methods: Based on the amyloid hypothesis, a 10 amino acids β-sheet breaker peptide HPYD (His-Lys-Gln-Leu-Pro-Phe-Tyr-Glu-Glu-Asp) was designed according to the structure and sequence of the previous designed peptide H102. Accelerated stability test, thioflavine T (ThT) fluorescence spectral analysis and transmission electron microscopy (TEM) imaging were performed to detect the stability and inhibitory effects on the aggregation of Aβ1−42 by H102 and HPYD. FITC-labeled HPYD was first tested to determine whether it could be transferred along the olfactory pathway to the brain after nasal administration to mice. Subsequently, the Morris Water Maze (MWM) test for behavioral analysis was used to investigate the learning and memory ability of APP/PS1 transgenic mice by HPYD. Immunohistochemistry and western blot analysis was performed to determine the role of HPYD on Aβ and APP protein levels. In addition, microarray analysis was used to evaluate the effect of HPYD on gene expression in AD mouse models.Results: Our in vitro results demonstrated that HPYD had enhanced stability and inhibitory effects on Aβ1−42 aggregation compared to H102. HPYD could be delivered into the brain through nasal administration and improved the learning and memory ability in APP/PS1 transgenic mouse models by reducing Aβ and APP protein levels. In addition, microarray analyses suggested that several genes related to the inflammatory pathway, AD and gluco-lipid metabolism were dysregulated and could be restored to almost normal levels after HPYD administration to mice.Conclusions: Our results demonstrated that HPYD could be a potential therapeutic drug candidate for the treatment of AD. Alzheimer's disease (AD) is a complex, severe neurodegenerative disorder of the central nervous system, which manifest as progressive cognitive decline and memory impairment in the elderly and presenium individuals. More than 44 million people are currently living with AD and the global health care cost in 2010 was about US$818 billion  are 39–42 amino acid peptide residues that are derived from putative intramembranous processing of amyloid precursor protein (APP) by γ-secretase/PS1 aspartyl protease Selkoe, . Only a 2+, which then results in rapid and massive neuronal cell death  to stimulate inflammatory responses  or protofibrils to be the predominant toxic forms of Aβ  in TgCRND8 mice  that can reduce amyloid load and cerebral damage and improve the learning and memory ability of AD animal models  are a class of compounds that are highly potent in ameliorating Aβ1−42. We also investigated the ability of HPYD to transverse into the brain through the olfactory pathway after nasal administration of fluorescein isothiocyanate (FITC)-labeled HPYD. The effect of HPYD on APP/PS1 transgenic mice behavior and the APP and Aβ expression in the brain were also investigated. Furthermore, we profiled the gene expression in normal mice (control group), APP/PS1 transgenic mice (model mice) and APP/PS1 transgenic mice treated with HPYD (HPYD group) using gene microarrays. Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to annotate their functions. Our findings not only has important implications for the potential treatment of AD using HPYD, but also provide insights to the mechanism of Aβ toxicity in AD patients and for the development of new therapeutic strategies for AD.In this study, we first compared the stability of H102 and HPYD using the accelerated stability test, and then performed inhibitory studies on the aggregation of AβNude mice , APP/PS1 mice  and C57/6J mice (weight: 25.5 to 28.6 g) were purchased from the institute of laboratory animal sciences, CAMS & PUMC . APP/PS1 mice have been previously demonstrated to form amyloid plaques, which have been approved by institute of laboratory animal sciences, CAMS and PUMC. All animal experiments were performed in accordance with the China Physiological Society “Guiding Principles in the Care and Use of Animals” approved by Tianjin Medical University Animal Care and Use Committee (NO. 20130021).H102, FITC-labeled HPYD and HPYD were synthesized using the Fmoc solid-phase synthesis method and purified by HPLC . The compounds were greater than 95% pure as measured by HPLC-MS. HPYD, a polypeptide comprising the amino acid (AA) sequence of His-Lys-Gln-Leu-Pro-Phe-Tyr-Glu-Glu-Asp, was dissolved in normal saline.0.5 mg of HPYD or H102 was dissolved in 1 mL of 0.2 mol/L phosphate buffer containing 100 mg/L of trypsin. The trypsin solution and polypeptide were mixed at a ratio of 1:4, and the solution was placed in a temperature controlled water bath shaker at 37°C for 0, 40, 100, 160, 220, 280, 340, and 400 min. The samples were then heated to 80°C for 10 min, and the stability of HPYD and H102 was detected at the different time points. High performance liquid chromatography (HPLC) analysis of HPYD and H102 was performed using a Phenomerex C18 column . The mobile phase consisted of solvent A, 0.1% TFA in acetonitrile and solvent B, 0.1% TFA in water at a ratio of 22.5:77.5 (v/v). The sample injection volume was 20 μL. The flow rate was 1 mL/min, and the detection wavelength was 220 nm.1−42 freeze-dried powder was dissolved in 50 mmol/L sodium phosphate buffer solution (pH = 7.4) to a concentration of 22.15 μmol/L, and H102 and HPYD were dissolved in the same PBS solution to a concentration of 88.60 μmol/L. Aβ1−42 solution was then mixed with H102 and HPYD respectively in equal volumes. The Aβ fibrils were grown at 37°C for 24 hrs. 10 μL solution from each group was then added to 990 μL of 3.0 μmol/L ThT solution and fluorescent intensity was measured using a VARIAN PTC-Au00-01058 fluorescence spectrophotometer  with an excitation wavelength of 453 nm and emission of 478–486 nm.Aβ1−42 samples (11.07 μmol/L 20 μL) were incubated in 37°C for 5 days, and the mixture of Aβ1−42 (22.15 μmol/L 10 μL) with HPYD (88.61 μmol/L 10 μL) or H102 (88.61 μmol/L 10 μL) were incubated under the same conditions for 5 days. Aliquots (5 μL) of each sample was spotted onto thin carbon substrates supported by carbon film on a 300 mesh copper grid for 15 min and blotted dried. The TEM grids were negatively stained with 2% uranyl acetate for 2 min and air dried. The samples were subsequently imaged using a Hitachi H-600 transmission electron microscope.The AβNude mice were anesthetized using 20% urethane (5 mL/kg), and then placed on the VFIS observation platform. Nude mice were then irradiated at 490 nm excitation without treatment, and then irradiated for 5, 15, and 30 min after nasal administration with FITC-HPYD (5.535 mg/kg). Thirty minutes after nasal administration with FITC-HPYD, the mice were euthanized and the hippocampus, cortex, olfactory bulb, heart, lung, liver, spleen and kidneys were removed. These organs were then irradiated at 490 nm excitation.n = 12 for each group). C57BL/6J mice with the same genetic background and age served as the normal controls. Treatment group received intranasal administration of HPYD saline solution , while the normal control group and model group were given with the same volume of saline for 30 days.The APP/PS1 transgenic mice were randomized into the model and HPYD treatment group  according to our previous report  were added and incubated overnight at 4°C. Biotin-labeled secondary antibody was added and incubated for 15 min at room temperature, followed by incubation with strept avidin-biotin complex (SABC) and DAB chromogenic reagent. Finally, the sections were counterstained with hematoxylin and observed under a microscope.At the end of the MWM test, the mice were euthanized and their brains were removed and rapidly placed on ice. The brain tissues were then fixed in 4% paraformaldehyde solution and waxed. Tissue sections were then dewaxed, and antigen retrieval was performed using boiling citrate buffer solution. This was followed by incubating the sections in 3% HWestern blot analysis was performed according to the detail procedures described previously. Primary Aβ antibody (1:200) was purchased from Abcam (USA) and anti-APP antibody (1:200) was purchased from Wuhan BOSTER Bio Company . The secondary goat anti-rabbit antibody was purchased from Sigma-Aldrich .P ≤ 0.05), and signaling pathway analysis was performed using the KEGG data software. Software Matlab 7.1 and java, was used to build and analyze the dynamic Gene networks, and network maps of the differentially expressed genes were constructed from the different states.Microarray hybridization was carried out by Shanghai GMINIX Biotech Limited Company (China) using the GeneChip® Mouse Gene 1.0 ST Array  with 770,317 probes. Briefly, total RNA was extracted from frozen brain tissues using the RNeasy mini kit  and used for cDNA synthesis using the cDNA synthesis kit . cDNA was labeled using the Gene Chip2 WT Terminal Labeling Kit , and then the labeled cDNA was hybridized to the mouse Gene chip at 45°C for 16 h. The Gene chip was then washed with wash solution A, wash solution B and deionized water, and stained using Cocktail 1 and Cocktail 2. The Gene Chip 2 Scanner 300 7G (Affymetrix Inc.) and the AGCC Scan Control software was used for date analysis. The gene ontology (GO) enrichment analysis was performed using the GOEAST software toolkit  with UltraSYBR Mixture purchased from Beijing ComWin Biotech Co., Ltd. . Gene transcript normalization was performed using housekeeping gene β-actin. All primers used for RT and qPCR are listed in Table P ≤ 0.05 was considered to be statistically significant .All experiments were performed at least three times. Data is presented as mean ± SD. The date of escape latency from MWM were analyzed by multivariate analysis of variance (ANOVA) and the other data were analyzed by one-way ANOVA, followed by Student-Newman-Keuls test. All the analysis was performed by SPSS statistical software . in vitro, accelerated stability test using trypsin was performed. After treatment with 100 mg/L trypsin at 37°C for 400 min, followed by heat treatment for 10 min at 80°C, the concentration of HPYD was 81.67% ± 0.42, while the concentration of H102 was only 41.07% ± 0.17, indicating that the stability of HPYD was better compared to H102  have been approved worldwide to treat AD (Cummings et al., Herba Epimedii Maxim, was demonstrated to inhibit Aβ production by reducing APP and BACE1 expression in APP/PS1 transgenic mice (Yan et al., The amyloid hypothesis is one of the most accepted mechanistic hypothesis for AD posits that dysregulated aggregation and accumulation of Aβ in the brain leads to nerve fiber entanglement and neuronal apoptosis (Hardy and Selkoe, in vitro. We designed alternative β-sheet breaker peptide, HPYD, by substituting Phe7 with Tyr7. This peptide showed higher in vitro stability and better efficacy of inhibiting Aβ aggregation compared to H102. In addition, FITC-labeled HPYD demonstrated that the peptide could enter the brain after nasal administration. MWM test demonstrated significant spatial learning and memory disorders in APP/PS1 transgenic model mice, which were in concordance with previous studies (Yan et al., Previously, we designed a β-sheet breaker peptide, H102, that could inhibit Aβ aggregation as well as reducing amyloid load in the brains of APP/PS1 transgenic mice, thus limiting brain damage and improving the symptoms of AD in animal models (He et al., 1−42 (Sagy-Bross et al., To further demonstrate that HPYD is a potential therapeutic for AD, we analyzed the mRNA levels of APP/PS1 transgenic mice to investigate the effect of HPYD on the brain. APP/PS1 transgenic model mice have early memory dysfunctions even before the degeneration of synapses and neurons (Dickey et al., in vitro stability and inhibitory effects on Aβ1−42 aggregation compared to H102. HPYD could improve the learning and memory ability in APP/PS1 transgenic mice by reducing Aβ and APP protein levels. Microarray analyses demonstrated that dysregulated gene expression in model mice could be restored to normal levels after treatment with HPYD by inhibiting the aggregation of Aβ. This provides a novel therapeutic strategy for the treatment of AD.In summary, we designed a new β-sheet breaker peptide HPYD, which showed better SX conceived project, WL, FS, and SX designed the study and wrote the paper. WL and FS performed the experiments. MW, FJ, XB, and LL provided technical assistance and contributed to the preparation of the figures and manuscript. HT contributed to English editing and academic writing in the whole manuscript. All authors reviewed the results and approved the final version of the manuscript.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.


Clinical pathways are widely used around the world for providing quality medical treatment and controlling healthcare cost. However, the expert-designed clinical pathways can hardly deal with the variances among hospitals and patients. It calls for more dynamic and adaptive process, which is derived from various clinical data. Topic-based clinical pathway mining is an effective approach to discover a concise process model. Through this approach, the latent topics found by latent Dirichlet allocation (LDA) represent the clinical goals. And process mining methods are used to extract the temporal relations between these topics. However, the topic quality is usually not desirable due to the low performance of the LDA in clinical data. In this paper, we incorporate topic assignment constraint and topic correlation limitation into the LDA to enhance the ability of discovering high-quality topics. Two real-world datasets are used to evaluate the proposed method. The results show that the topics discovered by our method are with higher coherence, informativeness, and coverage than the original LDA. These quality topics are suitable to represent the clinical goals. Also, we illustrate that our method is effective in generating a comprehensive topic-based clinical pathway model. Clinical pathway (CP) is of great importance in disease treatment, hospital management, and government regulation. It has been widely used to make the treatment process more standardized and normalized. The core principle of CP is to divide the clinical treatment into several stages and detail the clinical activities in each stage. Now, there are more than 300 CPs which have been designed by experts and implemented in China. Recently, clinical pathway mining (CPM) has experienced increased attention, which refers to using data mining technologies to discover the execution CP from hospital historical data. In comparison with the expert-designed CP, the data-driven execution CP is more objective. It can facilitate the CP (re)design, CP implementation, and abnormal detection.The most current CPM approaches are based on the process mining technologies, which are popular in extracting process models from event logs. The resultant graph models contain two basic elements: nodes represent clinical activities and edges represent the order relations between these activities. However, the clinical data are too unstructured and complex for process mining methods. They usually result in spaghetti-like models which are comprised by massive nodes and edges. To make the process model more comprehensive, researchers attempt to manually predefine the key activities for process mining. It is effective in promoting the model conciseness and highlighting core information, while the manual works are always expensive and subjective.Some other approaches focus on using topic modeling methods to discover clinical patterns instead of process models. They assume that the treatment for a disease can be concluded into a number of patterns. For a patient, a doctor would choose one pattern for him/her according to the symptoms. To achieve this target, topic modeling technologies, such as latent Dirichlet allocation (LDA)  and its The clinical activities occurring in a specific time duration  are prescribed around several clinical goals.Each clinical goal corresponds to a clinical activity set.In our previous work , we takeTake the first day of ICH as the example  In To address the above problems, we proposed a novel extension of LDA for CPM, which is called Clinical Daily Goal LDA (CDG-LDA). As shown in We incorporate topic assignment constraint into LDA to make the inference procedure consistent for the same clinical activities in one clinical day.Informativeness is introduced to adjust the ranking in each topic, which can improve the topic quality.Experiments on real-world datasets demonstrate the great performance of our approach for CPM by applying process mining on the quality discovered topics.Our main contributions in this paper are as follows:A clinical activity is an event occurring at a particular time point, which refers to the basic element in treatment.A patient trace contains a series of clinical activities. We segment each patient trace into a collection of clinical days, and a clinical day represents a set of clinical activities occurring in the same hospitalized day.We first give the definitions used in this paper:Early in 2001, Lin et al.  pointed Researchers attempted to adopt frequency-based methods to improve the ability of analyzing the complex process models. In , the patConsidering the complexity and quantity of clinical data, topic modeling technologies are used to discover the clinical patterns, which are useful in CP (re)design and abnormal detection. Huang et al.  firstly In this paper, we extend the work in  to enhanWe first introduce the conversion from billing data to the word-count format for LDA. Then, we present a brief review of applying LDA on clinical data. After analyzing the two shortages of the raw LDA, we incorporate topic assignment constraint and topic correlation limitation into LDA to improve the performance of discovering latent topics from clinical data.Billing data is the most common data recorded in hospital information system (an example is shown in the upper-left part of ϕk ~ Dir(β), k = 1, 2,…, KDraw distribution dth clinical day, d = 1, 2,…, Dθd ~ Dir(α)Draw distribution ith clinical activity in dth clinical day, i = 1, 2,…, Ndzd,i ~ Multi(θd)Draw ad,i ~ MultiDraw For For LDA is one of the most popular statistical topic modeling technologies. It models the generative process of each word from each document in a text dataset. There are two core model parameters in LDA: a corpus-level distribution over words for each topic and a document-level distribution over topics for each document. In clinical data, by regarding the clinical day and clinical activity as the document and word, respectively, the generative process of LDA has great conformity with the two abovementioned clinical practices. The graphical representation is shown in According to the graphical model, we need an inference to find the parameters which can best match the observed clinical data. Gibbs sampling, a special case of Markov chain Monte Carlo (MCMC), is the most popular inference method for LDA. It is based on the joint distribution of latent topics and observed clinical activities as follows:Given the joint distribution, the topic assignment for each clinical activity can be written as follows:ϕ and θ are computed as follows:ϕkv). So thaCdg is imposed to express the topic assignment constraint as follows:In this section, we adopt the chain graph proposed in  to incorCdg would equal to 1 only when all the clinical activities in the group get the same topic. Our goal is to make all the groups conform to the constraint. So that we add Cdg in the joint distribution of LDA , where zdg = k represents the set zdg which has the same topic k (. From (Nkv) in all clinical days. Due to the independence of the inference procedure of LDA, the frequency of v may be uniformly allocated to various topics. Thus, a high-frequency clinical activity is much easier to rank high in many topics, while a low-frequency clinical activity can hardly rank high in even one topic. It results in a large redundancy between different topics, so that each topic is lacking of characteristics. It is hard to regard these topics as the clinical goals.The ranking of a clinical activity v). From , it is oNv)( over topics. Higher informativeness and less relevant topicsRestrict the distribution of ϕkv) to measϕkv) = 3, its Nv) from 1 to K according to its IDF. Then, for each clinical activity value v, we maintain a corresponding relevant topic list κ(v). In every δ iteration, we check whether the size of κ(v) has been reduced to the desired number s(v) (line 12). To the clinical activities which still need to be restricted, we will update their lists (line 13) by discarding the topic with minimum relevant value (RV) as defined ink, v) is the ranking of clinical activity v in topic k based on the multinomial distribution ϕk. The numerator Nkv) reflects the importance of clinical activity v to topic k. Thus, higher RV represent more correlation between the clinical activity and the topic.The topic number upper bounder refers to the maximum number of the topics a clinical activity can correlate to. For example, given a clinical activity ECG and its topic number upper bounder K = 5 topics and a clinical activity ECG with a RV vector {50, 150, 5, 100, 60} and s(ECG) = 3, the relevant topic list k(ECG) would be changed from {1, 2, 3, 4, 5} to {1, 2, 4, 5} in the next updating procedure. As seen in line 15 of By keeping on updating the relevant topic lists, we gradually reduce the dimension of topic selection to the desired range. For instance, suppose there are zdg is 𝒪(K), where K is the number of topics. Therefore, the overall complexity is 𝒪(KδGK) = 𝒪(K2δG). δ is the number of iteration for updating relevant topic list and G = ∑dDGd. In practice, K would not be a large value and δ could be treated as a constant, so that G is the key factor that contributes to the complexity. It means that the proposed method scales linearly as we increase the total number of groups.The complexity of deriving a sample Instead of the detailed and complex clinical activities, we use the discovered topics to represent each clinical day. Hence, each patient can be regarded as a topic-based sequence. We adopt the process mining framework proposed in  to derivIn this section, we conducted a series of experiments to demonstrate the effectiveness of the proposed methods in discovering quality CP model. We begin with the description of experimental settings.α = 1.0 and β = 0.01.To evaluate both the suitability and generality of our approach, we collected two real-world billing datasets from a city-centre hospital. The two datasets are about two different diseases: an internal disease—intracerebral hemorrhage (ICH), and a surgical disease—inguinal hernia (IH). The detailed statistics are summarized in K is determined by a trade-off strategy proposed in [K, while perplexity is not strongly correlated to human judgement [K would significantly reduce the interpretability of the model. Thus, we select the intersection point of the two metrics across various K as the optimal topic number (K = 8 for ICH and K = 5 for IH). The iterations for updating relevant topic list δ are set to 300.The topic number posed in . It atteudgement . In our N = 10 ranked clinical activities and asked doctors to label it. The similar topics generated by different methods are in the same row. Note that, given a top size N, we defined 𝒯  as all the top N-ranked clinical activities among K topics. For example, 𝒯 refers to the 80 clinical activities for ICH when N = 10 and K = 8.The latent topics discovered by topic modeling are interpreted as the clinical goals in our approach. To evaluate the quality of the topics, we compare our approach (CDG-LDA) against LDA  in threeWe expect that the clinical activities in a topic could represent the similar clinical goal. Take topic 5 , relevant (score 1), or irrelevant (score 0). The final score is determined by a majority voting strategy. The kappa value, which is used to measure the interrater agreement, is 0.73 for ICH and 0.74 for IH. We used NKQM@N  as the m𝒯) is the unique clinical activities in 𝒯 and count(v) is the number of the occurrence of clinical activity v in the 𝒯 . In the previous example in N > 3). Lower RE represents less redundancy and hence a better clustering result.Given two discovered topics, we expect that they would not look alike, which means that the two topics should contain few same clinical activities. Thus, the redundancy indicator focuses on the distinctive characteristic of each topic. We measure the redundancy  by calcK and top size N. This is due to the incorporation of topic correlation limitation. By updating the relevant topic list during the iterations of Gibbs sampling, each clinical activity would strongly correlate to limited topics, especially the informative clinical activities. As a sequence, a clinical activity would not rank high in many topics, which lead to a low redundancy degree among all the topics.𝒯. We used the necessary/recommendatory clinical behaviors in the Chinese National Clinical Pathway and Chinese/AHA/ASA/EHS guideline as our benchmark. The overlapping degree between 𝒯 and the benchmark is adopted as the coverage indicator. Here, we took the 𝒯 listed in Tables N = 10, to analyze the coverage from three aspects:ExaminationCoverage refers to the ability of discovering important clinical activities for a specific disease. Given two high coherent and low redundant topics  for ICH, both of them are about the basic clinical equipment. However, these clinical activities are not critical enough for the treatment of ICH. We aim to find out all the key clinical activities in For ICH, the core examinations contains blood/urine routine, ECG, and biochemical and imaging examinations. For the first three, both LDA and CDG-LDA have discovered majority of the related clinical activities in topics 5, 6, 7, and 8. While for the last one, LDA failed to find out brain CT, which is the most effective imaging examination for ICH. In CDG-LDA, brain CT ranked high in topic 5.(ii) TreatmentFor IH, a patient needs the similar examinations to ICH except the imaging test. In topics 1 and 3 of the two methods, we can find the clinical activities about blood/urine routine, ECG, and biochemical examinations. An interesting observation is that the topics in the 2nd row got by LDA and CDG-LDA are quite different. In LDA, the activities in topic 1 are mainly about the infectious disease assay, which is an important part of biochemical examination. While topic 2 of CDG-LDA focuses on the presurgical preparation. Although LDA got better performance in infectious disease examinations, we deem that the result of CDG-LDA is more suitable for the clustering of IH clinical behaviors. There are two reasons: (1) The infectious disease assay-related activities can be found in the top 20 of topic 1 in CDG-LDA. They all belonged to the biochemical examinations which will always be prescribed together in practice, so that it is reasonable to put them in the same topic. (2) Topic 2 about presurgical preparation is of great importance for IH. We would detail this in the following part. Due to the ICH dataset is extracted from the Neurology Department rather than the Neurosurgery Department, the treatment activities are mainly medical instead of surgical. According to the drug function, the commonly used medication for ICH can be divided into four categories: reducing ICP (dehydration), keeping electrolyte balance, controlling blood pressure, and preventing complications . Most of them have been covered by both LDA and CDG-LDA. However, LDA missed an important dehydration drug piracetam, which is of high frequency in the dataset.(iii) Nursing careIH is a typical surgical disease, so that the treatment activities are mainly about the surgery. It is observed that both LDA and CDG-LDA contained the surgery-related clinical activities in topic 5. However, LDA failed to discover the most critical surgical activity IH repair and anesthesia in the top 10 of topic 5. This is caused by the high redundancy between topics 4 and 5 of the LDA. It made the ranking of these important surgical activities lower than the nursing care activities, which are of high-frequency. Moreover, as we discussed before, few of presurgical activities have been found by LDA. By limiting the topic number of each clinical activity and adjusting the day-activity distribution calculation, CDG-LDA successfully discovered these clinical activities in topic 4. In both ICH and IH datasets, the frequency of the clinical activities about nursing care are always the highest. Hence, the two topic modeling methods got good performance in discovering this kind of activities, including different level nursing care and various nursing equipment.(1) Discussion of Topic Quality. From the abovementioned three aspects about coherence, redundancy, and coverage, we can tell that CDG-LDA demonstrates significantly better performance than the original LDA in discovering latent topics from clinical data. The extracted high-quality topics can be suitably interpreted as the clinical goals.ICHBy using a topic label  to represent each clinical day, we got a series of topic-based sequences. In this section, we demonstrate the process models derived from these sequences by using the process mining methods proposed in . Note thAdmission (topic (5), topic ): the patient would accept a series of examinations for making a definite diagnosis, including brain CT, ECG, and biochemical assays. Note that imaging examinations are not required for all the traces. This is due to the fact that the clinical activities in our imaging topic (topic (1)), such as CTA and MRA, are mainly used as the auxiliary means for the diagnosis of ICH. Usually, the patients with severe symptoms or comorbidities need further imaging examinations.Treatment (topic (3), topic , topic (4), and topic (2)): After various examinations, the ICH patients would receive nursing care and take medications for recovery. Because of the urgency of ICH, drugs and high-level nursing care are firstly used to relieve and monitor the symptoms. When symptoms become stable, the nursing level may be changed to regular. As an internal department, various medications are used for ICH treatment according to the patients' symptoms.Re-examination (topic (5), topic , and topic (1)): During the treatment stage, related examinations are repeatedly needed for confirming the patients' states.IHThe process can be easily divided into three stages. Similar to ICH, the IH patients would firstly receive various examinations. Then, a series of presurgical activities are used for them. Note that some patients would have surgery in the same clinical day to the presurgical preparations ), while others may accept surgery in the next clinical day (topic (2)). The average level of nursing care for IH is lower than ICH. And the antibacterial drugs are used during the surgery and nursing care period. It is worth mentioning that the majority of IH patients have undergone the surgical treatment; while it still existed, a small number of IH patients have not received surgery. By careful analysis, we found that these patients are mainly the infants or the elders who are not appropriate for surgery.(1) Discussion of Process Model. It is observed that the topic-based clinical process model is of great conciseness and interpretability for CPM. We can conveniently draw the execution CP from the clinical historical data and identify the characteristics compared to expert-designed CP.In this paper, we proposed a novel topic modeling approach to discover latent topics as the clinical goals for CPM. The key idea is to incorporate clinical practice into the generative process of LDA to improve the topic quality. Topic assignment constraint restricts that all the same clinical activities in one clinical day should be assigned the same topic. Topic correlation limitation incorporates informativeness feature to adaptively adjust the ranking of the clinical activities in each topic. Process mining methods are used on these topic-based sequences instead of the detailed clinical activities. The experimental results show that our approach outperforms the original LDA in coherence, redundancy, and coverage. And the resultant topic-based process models are of great conciseness and interpretability for CP representation.One extension of this work is to find a more suitable measure as the informativeness indicator to improve the ranking adjustment strategy.The authors declare that there is no conflict of interest regarding the publication of this paper.


A total of 546 students participated in the Tier 1 Program of the P.A.T.H.S. Project responded to the Chinese Positive Youth Development Scale (CPYDS) at pretest and posttest and the Subjective Outcome Scale (SOS) at posttest. Result showed that the SOS was internally consistent. The SOS total scores were significantly related to measures of global satisfaction and the participants' degree of sharing with others, thus giving support to its construct validity. Factor analysis revealed that there were three dimensions of the scale and the related subscales were significantly correlated among themselves. Based on the significant relationships between the SOS measures of perceived program effectiveness and posttest CPYDS scores as well as changes in CPYDS scores, the present study revealed the convergence of subjective outcome evaluation findings and objective outcome evaluation findings in the P.A.T.H.S. Project.


EPM2A (laforin) or EPM2B . It characteristically involves the accumulation of insoluble glycogen-derived particles, named Lafora bodies (LBs), which are considered neurotoxic and causative of the disease. The pathogenesis of LD is therefore centred on the question of how insoluble LBs emerge from soluble glycogen. Recent data clearly show that an abnormal glycogen chain length distribution, but neither hyperphosphorylation nor impairment of general autophagy, strictly correlates with glycogen accumulation and the presence of LBs. This review summarizes results obtained with patients, mouse models, and cell lines and consolidates apparent paradoxes in the LD literature. Based on the growing body of evidence, it proposes that LD is predominantly caused by an impairment in chain-length regulation affecting only a small proportion of the cellular glycogen. A better grasp of LD pathogenesis will further develop our understanding of glycogen metabolism and structure. It will also facilitate the development of clinical interventions that appropriately target the underlying cause of LD.Lafora disease  is a rare, recessively inherited neurodegenerative disease with adolescent onset, resulting in progressive myoclonus epilepsy which is fatal usually within ten years of symptom onset. The disease is caused by loss-of-function mutations in either of the two genes  EPM2A) L [30]), L , the α aL , the proL , Hira-inL , and EPML . These pThe interaction of laforin with malin and their collaborative function is supported by a large number of studies ,53,57,58EPM2B , a single-exon gene located on chromosome 6p22.3, is a 395 amino acid protein that contains a RING and six NHL-repeat domains, the former strongly suggesting an E3 ubiquitin ligase activity [Human malin, encoded by activity ,60. As iactivity .There is evidence that the laforin-malin complex predominantly incorporates K63-linked polyubiquitin chains ,62,63. TMalin has been shown to ubiquitinate laforin, thereby promoting its degradation . FurtherIn summary, the function of polyubiquitination by the laforin-malin complex thus seems to link autophagy-related processes to glycogen metabolism. It therefore remains to be discussed how both processes could lead to LB accumulation.A mutated laforin that possesses a catalytically inactive phosphatase site is able to prevent LD in mice deficient of endogenous laforin . MoreoveIt may, however, be informative to summarize the current knowledge of (de)phosphorylation of another polyglucan-storing biological system, i.e., plant starch metabolism. Typically, plants form and mobilize water-insoluble starch granules as a functional equivalent to glycogen in animals . With thAnother factor that has been discussed as causative of LB accumulation is simply the cellular glycogen concentration. This hypothesis is supported by the fact that accumulation of insoluble glycogen (polyglucosan) is observed as a result of increased GS activity by overexpression of PTG or of GS itself ,39. WhilBy contrast, the abnormal chain length distribution of glycogen strictly correlates with the occurrence of LBs in laforin- and malin-deficient mice . The relThus, it is obvious that frequent and regularly distributed branching points lead to shorter chains and less double-helix formation. Indeed, a strong correlation of chain length distribution and polyglucan crystallinity has been demonstrated using a range of mutant starches. In these studies, a high abundance of short glucan chains was consistently found to interfere with polyglucan crystallinity ,89. FurtA clear difference between water-soluble normal glycogen and the partially insoluble LD glycogen is the enrichment of longer α-glucan chains in the latter, as seen by its abnormal chain length distribution . SimilarIt should be noted that the chain length distribution has to date been determined as an average of all glycogen molecules present in the respective tissue sample. At a subcellular/submolecular level, minor differences in the activities of GS and GBE may be part of normal glycogen metabolism. Such localized differences could lead to variations of chain length distributions in individual molecules and/or even in some regions of a given glycogen molecule, but remain undetected by conventional chain length distribution analyses. There is, however, evidence that even in wild-type mice some glycogen molecules possess longer chains than others, i.e., that not all molecules within one cell have an identical chain length distribution ,92. It iWith a loss-of-function mutation in either laforin or malin causing essentially the same pathological phenotype, and the strong evidence for in vivo laforin-malin interaction ,53,68,93While plants possess the capacity to mobilize water-insoluble polyglucans (starch) under a wide range of external conditions, this is certainly not the case in animals, which generally have evolved to use water-soluble polyglucans (glycogen). Insolubility and aggregation of glycogen can have severe consequences in mammalian cells which becomes obvious in the neurons of LD patients. Thus, it is likely that hypothetical mechanisms to dispose of insoluble glucan material work at a low rate and may practically render precipitated glycogen metabolically inert.Avoiding precipitation altogether would be biologically important. One possibility is that the laforin-malin complex is able to decrease GS activity selectively on molecules that are likely to precipitate, for example molecules that are still soluble but are, on average or in particular molecule regions, composed of longer glucan chains A. IndeedHowever, a mechanism for the removal of abnormal and insoluble glycogen cannot be ruled out and may also involve a functional laforin-malin complex. One possible mechanism of removal could proceed via autophagy which has been shown to be impaired in some LD mouse models but not in others ,38,51. TThus, the function of the laforin-malin complex may be implicated in (1) a locally focussed alleviation of glycogen precipitation risks, (2) a selective removal of abnormally formed and precipitated glycogen, or (3) in both. All of these hypotheses are unified in the assumption of an underlying intrinsic property of glycogen metabolism to inadvertently produce disadvantageous structural variations of glycogen molecules including some that are at risk to precipitate. The hypotheses differ in how they assume the cell copes with the risk of glycogen precipitation. While according to the first hypothesis, glycogen precipitation is avoided as much as possible, the second hypothesis implies a removal of abnormal and precipitated glycogen by autophagy. It is not inconceivable that mammalian cells employ both of these strategies at the same time (hypothesis 3,


Epoxyeicosatrienoic acids (EETs) are produced by cytochrome P450 epoxygenases from arachidonic acid, and their rapid metabolism is mainly through soluble epoxide hydrolase (sEH). EETs exert vasodilatory, anti-inflammatory, anti-apoptotic, and pro-angiogenic effects. Administration of sEH inhibitors before or at the onset of stroke is protective, but the effects of post-treatment at reperfusion, when inflammation is augmented, has not been as well studied. We tested the hypothesis that 1-Trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl)urea (TPPU), a potent and highly selective sEH inhibitor, suppresses inflammation and protects the brain when administered at reperfusion. Vehicle or 1 mg/kg TPPU was administered at reperfusion after 90 minutes of focal ischemia and again 24 hours later. Protein expression and activity of sEH increased after reperfusion and activity was decreased by TPPU administration. TPPU decreased infarct volume by 50%, reduced neurologic deficits and improved performance on sensorimotor tasks. Furthermore, TPPU significantly lowered the mRNA expression of interleukin-1beta by 3.5-fold and tumor necrosis factor-alpha by 2.2-fold, increased transforming growth factor-beta mRNA by 1.8-fold, and augmented immunostaining of vascular endothelial growth factor in peri-infarct cortex. Thus, inhibition of sEH at reperfusion significantly reduces infarction and improves sensorimotor function, possibly by suppressing early proinflammatory cytokines and promoting reparative cytokines and growth factors. Thrombolysis with tissue plasminogen activator and endovascular thrombectomy are currently the major treatments for patients with acute ischemic stroke. However, for some patients, reperfusion after thrombolysis and thrombectomy could exacerbate the injury by triggering multiple pathologic processes, including the inflammatory cascade, lipid peroxidation, mitochondrial dysfunction, and disruption of the blood-brain barrier. Therefore, identification of agents that target multiple mechanisms during reperfusion would be beneficial for limiting progressive neuronal cell death and promoting an environment that facilitates brain repair.5. In various organs, EETs exert broadly protective effects, including anti-apoptotic, anti-inflammatory, vasodilatory, anti-nociceptive, anti-epileptic, and pro-angiogenic effects14. However, EETs are rapidly hydrolyzed by soluble epoxide hydrolase (sEH) into less bioactive 1,2-diols, dihydroxyeicosatrienoic acids (DHETs)15. The sEH enzyme is broadly distributed throughout the central nervous system, with cellular expression in astrocytes, neurons, and vascular endothelium17. Gene deletion of sEH has been shown to reduce infarct volume after transient middle cerebral artery occlusion (MCAO) in male mice19 and reproductively senescent female mice20. However, sEH male knockout show a smaller decrease in cerebral blood flow during MCAO, thereby making it difficult to discern direct neuronal protection from effects of a less severe insult. Inhibitors of sEH have also been shown to reduce stroke infarct volume in mice and rats, stroke-prone spontaneously hypertensive male rats, and diabetic male mice24, but the inhibitors were administered as a pretreatment or at the onset of MCAO. With the success of endovascular thrombectomy in establishing recanalization and reperfusion in selected subpopulations of stroke patients, use of neuroprotective agents at the time of reperfusion is of clinical relevance. With regard to sEH inhibitors, the few studies of treatment at reperfusion have focused only on infarct volume as an endpoint20. Neurobehavior testing was not reported in these studies. Thus, limited data exists on the effect of sEH inhibitor administration at reperfusion and its effects on reperfusion injury.Epoxyeicosatrienoic acids (EETs) are lipid metabolites produced from arachidonic acid by cytochrome P450 (CYP) epoxygenases. In brain, EETs are predominantly generated by subfamilies of CYP2C and CYP2J, which are expressed in astrocytes as well as vascular endothelium25. EETs have been recognized as possessing anti-inflammatory properties in a variety of settings, such as lipopolysaccharide-induced inflammation27 and neuropathic pain28. However, under the conditions of cerebral ischemia, the effect of sEH inhibitors on cerebral cytokine expression is not clear-cut. Administration of an sEH inhibitor before MCAO or at reperfusion failed to attenuate cerebral induction of several proinflammatory cytokines29, whereas continuous intraventricular infusion of an inhibitor attenuated expression of inducible nitric oxide synthase23. In a model of cardiac arrest, administration of an sEH inhibitor after resuscitation failed to attenuate expression of proinflammatory interleukin-1β (IL-1β) or tumor necrosis factor-α (TNF-α)30.Neuroinflammation is an important component of reperfusion injury. Early release of proinflammatory cytokines is generally believed to contribute to the spread of infarction, whereas delayed release of anti-inflammatory cytokines contributes to the resolution of the infarction and the initiation of reparative mechanisms33 than many of the inhibitors previously used in stroke models. It also is taken up in rodent brain34. We tested the hypothesis that systemic administration of TPPU starting at reperfusion after MCAO in male rats reduces infarct volume, improves sensorimotor functional outcome, suppresses expression of proinflammatory IL-1β and TNF-α, augments expression of anti-inflammatory IL-10 and transforming growth factor-β (TGF-β), and decreases the number of peri-infarct microglia.The main objectives of the present study were to better characterize the response to administration of an sEH inhibitor at the time of reperfusion by examining effects on tissue cytokine responses, microglia number, and neurobehavior, in addition to infarct volume. Moreover, we used the sEH inhibitor 1-(1-propanoylpiperidin-4-yl)−3-[4-(trifluoromethoxy)phenyl]urea (TPPU). This newer generation sEH inhibitor possesses higher potency and a longer circulatory half-life17, but less information is available in rat cerebral cortex16. In non-ischemic rat cortex, sEH immunoreactivity was observed to be widely distributed , although activity remained slightly elevated relative to the sham value. Thus, TPPU produced the intended effect of limiting sEH activity.We then assessed the enzymatic activity of sEH activity in brain parenchyma by measuring the rate of conversion of 14,15-EET to 14,15-DHET with ELISA. Compared with that of the sham group, sEH activity significantly increased 1 day after MCAO in the vehicle group Fig. , indicatn = 12 rats per group). In the vehicle group, the median and interquartile range (IQR) of infarct volume in cortex, striatum, and hemisphere were 24% , 75% , and 23% , respectively , 57% , and 8% . Thus, post-treatment with TPPU markedly decreased infarct volume.Rats were subjected to 90 min of MCAO, and then 1 mg/kg TPPU or vehicle was administered at reperfusion and again 24 h later. Infarct volume was measured at 48 h after reperfusion. Statistical comparisons between the two groups were made by Mann-Whitney test . The lack of a greater mortality after TPPU treatment indicates that the smaller infarct volume seen with TPPU was unlikely due to greater attrition of rats with worse injury.When pooling rats with vehicle treatment from these and other outcome measurement cohorts, nine rats died within 24 h after reperfusion, and three died between 24 and 48 h, resulting in a mortality rate of 16.7% (12/72). With TPPU treatment, in contrast, only two rats died within 24 h, and one died between 24 and 48 h after reperfusion, resulting in a mortality rate of 4.8% (3/63). This trend of mortality rate was not statistically different . In addition, the modified sticky tape test was used to assess sensory deficits. Attention to the affected forelimb relative to the unaffected limb was decreased in the vehicle group compared to that in the sham group  and not significantly different from that in the sham group. We also performed a foot-fault test during walking on a grid. The difference between the contralateral and ipsilateral foot faults was greater in the vehicle group than in the sham group .At 2 days of recovery, we performed a neurologic assessment as described in Supplementary Table oup Fig. . This raoup Fig. , but thip < 0.01; n = 5 rats per group).To assess the effects of TPPU on neuronal cell death in the peri-infarct areas of cortex, we double stained with terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and the neuronal marker NeuN. Staining revealed an obvious increase in the TUNEL-positive neurons at day 2 after the ischemic insult, in contrast with the sparse distribution in sham groups Fig. . TPPU mip < 0.05; n = 6 rats per group). Expression of TGF-β mRNA also was elevated on days 1 and 2 after MCAO in the vehicle group and was increased even further on day 2 in the TPPU group (p < 0.05 vs. vehicle group). However, IL-10 mRNA did not differ significantly between the vehicle and TPPU groups.Inflammatory cytokines play a major part in the development of secondary injury after reperfusion, mediating the recruitment of inflammatory cells to the ischemic lesion. We determined the expression of proinflammatory cytokines IL-1β and TNF-α and anti-inflammatory factors IL-10 and TGF-β by both real-time PCR and Western blot analysis or ELISA. As shown in Fig. n = 6 rats per group). With TPPU treatment, TNF-α remained significantly elevated relative to the sham group on day 1, but was no longer significantly different from the sham level on day 2. TPPU treatment significantly increased TGF-β on day 2 relative to both the sham and vehicle groups . No significant changes in IL-10 protein were detected in either MCAO group relative to the sham group. Thus, TPPU treatment generally limited expression of proinflammatory cytokines and augmented expression of at least one of the anti-inflammatory cytokines.Using Western blot analysis and ELISA, we obtained results that generally paralleled the mRNA results Fig. . Proteinn = 5 rats per group), which exhibited relatively less cell body hypertrophy.We evaluated the number and morphologic characteristics of microglia after ischemia by immunolabeling them with Iba1. In the sham group, microglia were typically resting cells with small cell bodies and long, ramified processes Fig. . At day 35, which is consistent with the ability of EETs to promote VEGF release and tissue repair in a variety of organs13. Because VEGF promotes both neuronal protection and reparative mechanisms, we investigated the effect of TPPU on VEGF expression. We performed double immunostaining for VEGF with the NeuN or with the astrocyte marker GFAP on day 2 after MCAO . As expected, GFAP+ cells displayed relatively larger cell bodies and thicker processes, typical of astrogliosis after ischemia. The percentage of VEGF+-GFAP+ cells increased significantly by 36% and 57% in the vehicle and TPPU groups, respectively, relative to that in the sham group. This increase was significantly greater in the TPPU group than in the vehicle group (p < 0.001). These data indicate a differential regulation of VEGF expression after MCAO between neurons and astrocytes and that TPPU treatment after reperfusion can increase the number of both neurons and astrocytes expressing VEGF.Previous work showed that application of TPPU to cultured astrocytes after oxygen-glucose deprivation increases VEGF expression and releaseCAO Fig. . In the In this study, we investigated whether administration of the sEH inhibitor TPPU at reperfusion after transient focal ischemia provides neuroprotective effects and explored possible mechanisms for this protection. The major findings of this study are that post-treatment with TPPU (1) suppresses brain sEH activity and significantly decreases infarct volume in both cortex and striatum, (2) significantly improves motor and sensory functions, (3) attenuates peri-infarct cortical neuron cell death, (4) effectively reduces expression of pro-inflammatory cytokines IL-1β and TNF-α and augments anti-inflammatory TGF-β during the acute phase of recovery, (5) decreases the number of peri-infarct microglia, and (6) promotes astrocyte expression of VEGF at 48 h after reperfusion.24. One study in mice reported a decrease in infarct volume when sEH inhibitor administration was delayed until reperfusion8. Our current finding that infarct volume was decreased when TPPU administration was delayed until reperfusion in rats is consistent with the concept that sEH inhibitors can act independently of effects on collateral blood flow during the period of MCAO. Moreover, our data extend previous reports by demonstrating that delaying sEH inhibition until after reperfusion can improve performance on a neurologic exam, the sticky tape sensory test, and a foot-fault sensorimotor test. Efficacy of sEH inhibitor administration at reperfusion may have clinical significance for adjunct therapy with thrombolysis and thrombectomy. However, a limitation of the present study is that we did not study aged animals or animals with cardiovascular co-morbidities. It will be important to test TPPU administration at reperfusion in aged rats of both sexes and with comorbidities for evaluating the full translation potential of this target.Several studies of sEH inhibitors administered before or at the onset of MCAO in male mice and rats have confirmed a decrease in infarct volume, but without detectable changes in intraischemic perfusion, suggesting other mechanisms of action16. We did not explore whether the upregulation of sEH was limited to specific cell types, but others have recently reported enhanced sEH staining associated with astrogliosis after MCAO23. Our results show that the 1 mg/kg dosing of TPPU was sufficient to suppress the post-ischemic increase in brain sEH activity. We chose to use TPPU because it has high potency for inhibiting sEH, and a longer plasma half-life than many of the earlier generation of sEH inhibitors32. It also has greater water solubility, less binding to plasma proteins, and an extended resident time on the sEH molecule. Importantly for neuroprotection studies, it crosses an intact blood-brain barrier in rodents.Interestingly, we observed an increase in sEH expression and enzymatic activity after reperfusion. In non-ischemic brain, we observed sEH immunoreactivity in astrocytes and neurons, and others have also shown expression of sEH in blood vessels36. When we analyzed the expression of inflammatory factors, we found that TPPU administration decreased proinflammatory IL-1β and TNF-α expression but augmented anti-inflammatory TGF-β expression. This finding that an sEH inhibitor can limit expression of proinflammatory cytokines is consistent with a report showing decreased expression of inducible nitric oxide synthase after MCAO in rats administered an sEH inhibitor intraventricularly23. The latter finding suggests that the anti-inflammatory effect of an sEH inhibitor after MCAO can be attributed to actions within the brain and not necessarily by actions on the peripheral immune response. Also, consistent with a role for EETs in suppressing inflammation in brain after MCAO, overexpression of endothelial CYP enzymes that produce EETs limits the inflammatory response after MCAO14. After cardiac arrest, administration of an sEH inhibitor was reported to increase IL-10 induction in brain30. Unexpectedly, we failed to find an increase in IL-10 protein at 1 or 2 days of reperfusion with either vehicle or TPPU treatment in our focal stroke model. Perhaps longer recovery periods would be required to see an effect on this cytokine associated with infarct resolution and repair. Investigation of an extended panel of cytokines and chemokines at multiple time points is warranted in future studies to better characterize the effect of sEH inhibiton on the dynamics of the neuroinflammatory response.Based on these results, we investigated the factors that might contribute to the neuroprotective effects of post-ischemic TPPU administration. During the acute phase of ischemic stroke, microglia are activated rapidly, and inflammatory cytokines and chemokines are secreted largely by microglia and other cells to orchestrate the inflammatory response23. It should be noted that the decrease in microglia number in our study was associated with relatively similar attenuation of TUNEL-positive neurons. Based on our data, we cannot distinguish whether TPPU’s effect on inflammation caused a decrease in neuronal cell death, or if a primary effect of TPPU on neuronal cell viability reduced the inflammatory response. Alternative mechanisms whereby TPPU exerts neuroprotection are by the inhibitory effect of EETs on reactive oxygen species generation, mitochondrial dysfunction37, and apoptotic signaling5 and by the vasodilatory effects of EETs39. A limitation of the present study is that we did not investigate oxidative stress, mitochondrial dysfunction, apoptotic signaling, or reperfusion defects in the microcirculation. In cell culture experiments, EETs and sEH inhibitors are capable of directly protecting primary cultured neurons and astrocytes from oxygen deprivation22, thereby indicating the potential for actions independent of immune cells and perfusion defects. Because of the low basal expression of sEH that we observed in microglia, reduced inflammation and the inhibition of microglial activation with the sEH inhibitor may be mediated indirectly by regulation of astrogliosis and attenuation of neuronal apoptosis23.An anti-inflammatory effect of TPPU is also supported by our observation that the normal increase in number of microglia in the peri-infarct cortex was attenuated by administration of TPPU. A similar attenuation has recently been reported by others with a different inhibitor41. It also promotes angiogenesis42 and regeneration13. We immunostained sections of rat brain for the combination of VEGF and NeuN or GFAP. In the sham group, VEGF was predominantly associated with neurons and to a lesser extent with astrocytes. However, cell counts suggested that inhibition of sEH potently promoted astrocyte-derived VEGF in the cortical peri-infarct region. In the vehicle group, VEGF-positive neurons decreased markedly after MCAO and reperfusion. This decrease is likely due, in part, to the injury to neurons after ischemia and reperfusion. Furthermore, the increase in the number of VEGF-positive neurons after TPPU treatment may be secondary to the rescue of viable neurons, as supported by the decrease in TUNEL staining. Previous work in cell culture demonstrated that after oxygen-glucose deprivation, inhibition of sEH with TPPU dramatically increases astrocytic release of VEGF into the medium and that exposure of neurons to the astrocyte-conditioned medium rescued them from oxygen-glucose deprivation through VEGF receptor 2 signaling35. Another study reported that application of 14,15-EET directly to astrocytes after oxygen-glucose deprivation increases the release of brain-derived neurotrophic factor43. Therefore, one mechanism by which TPPU may exert protection after ischemia is through augmentation of trophic factor release from astrocytes. We speculate that the increase in trophic factor release by sEH inhibition could have a clinically relevant therapeutic window for brain repair and functional outcome.Our immunoreactivity results showed that sEH is well expressed in astrocytes. Astrocytes are the most abundant cells in brain, and one of their important functions is to release neurotrophic factors. VEGF is a trophic factor that protects neurons directly through Akt and ERK signaling pathwaysIn summary, our study provides support that targeting sEH specifically during the post-ischemic reperfusion period is beneficial. Daily administration of the potent sEH inhibitor TPPU reduced infarct volume and peri-infarct cell death, attenuated the neuroinflammatory response, promoted astrocytic VEGF expression, and improved performance of rats on sensorimotor tasks. Additional work is warranted to assess the therapeutic window for trophic factor release and the consequences for long-term functional outcome.44. After the right common carotid artery was exposed and occluded through a lateral incision, the external carotid artery and the proximal pterygopalatine artery were ligated. A 4–0 nylon suture with a rounded tip was inserted into the internal carotid artery and maintained there for 90 min before reperfusion. At least a 60% reduction of blood flow was verified by laser-Doppler flowmetry. In rats that underwent sham surgery, the arteries were isolated but not ligated, and no filament was inserted. Temperature was maintained with a heating lamp at approximately 37 °C throughout the surgery and early reperfusion. Rats were randomly assigned to a sham surgery group or to treatment with either vehicle or TPPU after 90 min of MCAO. TPPU was completely dissolved in polyethylene glycol 400 and was administered by intraperitoneal injection in an aqueous solution of 10% (vol/vol) normal saline at a dose of 1 mg/kg45 at reperfusion and again 24 h later.This study was approved by the Institutional Animal Care and Use Committee at the Johns Hopkins University, and all procedures on animals followed the approved protocol. Adult male Sprague-Dawley rats weighing 250–300 g were obtained from Charles River Laboratories . Anesthesia was induced in the rats with 5% isoflurane and maintained with 2% isoflurane during the surgery. MCAO was carried out with the intraluminal filament technique as previously described46 and is described in Supplementary Table 47 and uses a non-removable tape sleeve wrapped around the forepaw The time that the rat spent attending to this stimulus was recorded over a 30-s observation period. Outcome performance was expressed as a ratio of left attention time/right attention time. For the foot-fault test, the rats were placed on a horizontal metal grid suspended over a mirror, which aided us in detecting foot slips during video playback. A foot-fault was defined as a forelimb or hindlimb missing or slipping off the grid. We counted the number of foot-faults for the limbs contralateral and ipsilateral to the ischemic injury as well as the total number of steps taken over a 5-min period48. The results were calculated as the percentage of contralateral limb foot-faults per limb step minus ipsilateral limb foot-faults per limb step. Investigators blinded to treatment group assessed neurobehavioral performance and evaluated the results.At 2 days of recovery, we performed a neurologic examination, a modified sticky tape test, and a foot-fault test. The neurologic examination was based on a modified neurological severity scoring protocol44. The infarcted area was traced and analyzed with Image J software  by a person blinded to treatment group.At 48 h after reperfusion, the brain was harvested for measurement of infarct volume. Seven coronal sections (2-mm thick) were stained with a 1% solution of triphenyltetrazolium chloride (TTC)g at 4 °C for 30 min. We collected the supernatants for protein quantification with the bicinchoninic acid assay. Equal amounts of each protein sample were separated by electrophoresis in 4–15% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene fluoride membranes. After being blocked, membranes were probed during overnight incubation at 4 °C with the following primary antibodies: mouse anti-β-actin , goat anti-sEH , rabbit anti-IL-1β , rabbit anti-IL-10 , and rabbit anti-TGF-β . We quantitatively analyzed the protein bands with Image J software to obtain the optical densities.Brains were collected at 24 or 48 h after surgery for Western blotting. The tissues from the ipsilateral cortex were homogenized in RIPA buffer  with protease inhibitor cocktail  and then centrifuged at 10,000 ® Reagent  by following the manufacturer’s protocol. The first-strand cDNA was synthesized by High Capacity RNA-to-cDNA Kit . Quantitative real time-PCR was performed with Power SYBR@Green PCR Master Mix (Applied Biosystems) on an ABI 7500 FAST Real-Time PCR System (Applied Biosystems). The sequences of primers were as follows: forward 5′- CACCTCTCAAGCAGAGCACAG, reverse 5′-GGGTTCCATGGTGAAGTCAAC for IL-1 β; forward 5′-AGATGTGGAACTGGCAGAGG, reverse 5′-CCCATTTGGGAACTTCTCCT for TNF-α; forward 5′-CCTGCTCTTACTGGCTGGAG, reverse 5′-TGTCCAGCTGGTCCTTCTTT for IL-10; and forward 5′-AGATGTGGAACTGGCAGAGG, reverse 5′-CCCATTTGGGAACTTCTCCT for TGF-β. Expression levels of the target gene relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA were determined with the ΔΔCT method.We extracted total RNA from brain cortex with TrizolRat TNF-α levels in brain cortex tissue were measured by ELISA kits  according to the manufacturers’ protocols. Activity of sEH in rat brain cortex was measured by incubating brain homogenate with 14,15-EET for 1 h and assaying for 14,15-DHET with an ELISA kit . The optical density values were read on a plate reader  and normalized for protein concentration.At 48 h after reperfusion, the rats were deeply anesthetized and perfused transcardially with phosphate-buffered saline (PBS) and 4% paraformaldehyde. The brains were fixed overnight in 4% paraformaldehyde and immersed in 30% sucrose. Frozen 25-μm coronal sections were immunostained. In brief, after being washed three times in PBS, sections were blocked by 5% serum for 1 h. The samples were then incubated with appropriate primary antibodies: GFAP , NeuN , Iba1 , sEH , VEGF  and respective secondary antibodies  in PBS with 5% serum. Images were acquired on a fluorescent microscope . Five sections per rat brain were analyzed blindly by counting immunopositive cells in five fields adjacent to the ischemic lesion in cortex per section  with the aid of Image J software . For statistical analysis, the average value from 25 fields was used to obtain a single value for each rat.In Situ Cell Death Detection Kit, POD  according to the manufacturer’s instructions. Cells double stained with both NeuN and TUNEL were considered to be nonviable neurons. Their total number was obtained in a blinded fashion under light microscopy . Finally, an average for each group was calculated.Nonviable neuronal cells were detected by fluorescence with the Data were analyzed by analysis of variance, and post hoc comparisons were made with the Holm-Sidak procedure. For real-time PCR data, we performed a logarithmic transformation to allow the data to pass the equal variance and normality tests. Because infarct volume, neurobehavioral data, and protein levels did not pass the normality test or equal variance test, we used nonparametric tests. For infarct volume, differences between the vehicle and TPPU groups were analyzed with the Mann-Whitney Rank Sum Test. For neurobehavioral data and protein-level data, the Kruskal-Wallis analysis of ranks test was used. When there was an overall significant effect, pairwise comparisons were made with the Mann-Whitney test. A significance level of 0.05 was used in all tests. All results were expressed as the mean ± SD or as medians, quartiles, and 95% intervals.Original data will be provided upon request of the corresponding author.Supplementary Information