diff --git "a/deduped/dedup_0450.jsonl" "b/deduped/dedup_0450.jsonl"
new file mode 100644--- /dev/null
+++ "b/deduped/dedup_0450.jsonl"
@@ -0,0 +1,37 @@
+{"text": "Advances in electronic technology have created opportunities for new instructional designs of medical curricula.We created and evaluated a 4-week online elective course for medical students to teach the cognitive basis for interviewing skills.Ten students, from 2 medical schools, studied online modules on interviewing concepts and viewed videos illustrating the concepts. They then participated in asynchronous discussion groups designed to reinforce course concepts, stimulate reflective learning, and promote peer learning.In qualitative evaluations, learners reported improvements in self-awareness; increased understanding of interviewing concepts; and benefits of online learning vs face to face learning. Participants reported high levels of satisfaction with online learning and with achievement of course objectives. Self-reported knowledge scores increased significantly from pre-course completion to post-course completion.Online education has significant potential to augment curriculum on the medical interview, particularly among students trained in community settings geographically distant from their academic medical center. A number of organizations - 3 have The instructional design we use for online courses  has the Blackboard, a web-based learning system  was usedStudents received access to a moderated, asynchronous discussion board and were required to post their impressions and observations each week. If necessary , they were reminded by the moderator. Using established principles - 9 .Eleven formative evaluation questions  were preStudents also completed pre-course and post-course Web-based questionnaires with 21 items , each scr= 0.9412, P< .001), providing support for the construct validity of the self-reported knowledge measures  and opportunities to apply the concepts to real patients (Theme 4).Open-ended comments on the course evaluation form supported these themes, and provided more detail about advantages of online learning in this course over more conventional methods. Two students provided representative viewpoints:Student 1: \". . . interacting with students in the on-line format allowed for well thought-out, comprehensive responses and much more insightful comments than sometimes heard in a classroom. I attribute this to the time one has to sit and think through a response, choose the words carefully, and elaborate uninterrupted. There's less pressure on-line, so you can piece together your thoughts with less stress and greater sincerity.\"Student 2: \"The strengths are the high level of participation and interaction and conversation (more so than in any other course so far.)\"P< .01) at the end of the course   .r= 0.72, P=.02), suggesting that greater educational effort was correlated with greater self-reported gain in knowledge , in a speech once exhorted medical educators to seize \"the potential of the technological revolution to transform the way students learn\" . In resp"}
+{"text": "To report about initial clinical experience in radiation treatment of carcinoma of prostate with volumetric modulated arcs with the RapidArc (RA) technology.Forty-five patients with a median age of 72 \u00b1 3, affected by prostate carcinoma , with initial PSA of 10.0 \u00b1 3.0 ng/mL, were treated with RapidArc in a feasibility study. All patients were treated with single arc using 6MV photons. Dose prescription ranged between 76 (7 patients) and 78 Gy (38 patients) in 2Gy/fraction. Plan quality was assessed by means of Dose Volume Histogram (DVH) analysis. Technical parameters of arcs and pre-treatment quality assurance results  are reported to describe delivery features. Early toxicity was scored  at the end of treatment together with biochemical outcome (PSA).95% was in average higher than 98% and V107%~0.0% (D2%~104.0% in average). Homogeneity D5%-D95% ranged between 6.2 \u00b1 1.0% to 6.7 \u00b1 1.3%. For rectum, all planning objectives were largely met (e.g. V70Gy = 10.7 \u00b1 5.5% against an objective of < 25%) similarly for bladder (e.g. D2% = 79.4 \u00b1 1.2Gy against an objective of 80.0Gy). Maximum dose to femurs was D2% = 36.7 \u00b1 5.4Gy against an objective of 47Gy. Monitor Units resulted: MU/Gy = 239 \u00b1 37. Average beam on time was 1.24 \u00b1 0.0 minutes. Pre-treatment GAI resulted in 98.1 \u00b1 1.1%. Clinical data were recorded as PSA at 6 weeks after RT, with median values of 0.4 \u00b1 0.4 ng/mL. Concerning acute toxicity, no patient showed grade 2-3 rectal toxicity; 5/42 (12%) patients experienced grade 2 dysuria; 18/41 (44%) patients preserved complete or partial erectile function.From DVH data, target coverage was fulfilling planning objectives: VRapidArc proved to be a safe, qualitative and advantageous treatment modality for prostate cancer. In Switzerland an increasing incidence of prostate adenocarcinoma was observed in the last 10 years, with 5668 new cases/year, attaining to the 29.6% of all male malignancies in 2006, and an yearly average mortality of 1292 patients between 2003 and 2006 over a population of about 7.4 million inhabitants [A proper planning policy, which allows to spare the healthy tissue and at the same time ensure high cure rate, is of particular importance due to the rate of curability of this tumour and long survival of the patients. In this respect new, highly conformal treatments have been tested in the last years.Volumetric Modulated Arc Therapy (VMAT), based on the original investigation of K. Otto  has beenPre-clinical validation of RapidArc was addressed in a series of studies including brain tumours, head and neck, anal canal, cervix uteri cancer and other indications -9. The pAt our institute, until end of January 2010, more than 250 patients have been treated with RapidArc for a variety of indications. Among these, 117 received RapidArc treatment as part of their multidisciplinary management of prostate adenocarcinoma.Of these, forty-five, irradiated without inclusion of the pelvic nodes, were included in the present study based on the risk class. After a short transition time in the first weeks, all prostate patients are currently treated with RapidArc at our institute.Aim of the present study is to report the technical and dosimetric aspects of the treatments as well as to summarize the acute toxicity findings.Further investigations will aim to look at the long term clinical outcome and late toxicity in relation to dosimetric improvements in sparing of the organs at risk.Forty-five patients were treated with RapidArc (RA) from October 2008 to September 2009. Characteristics of patients are summarized in table The issue of target definition is highly debated for prostate cancer, particularly the inclusion of the seminal vesicles -18. AccoPatients were divided into two groups: Group A (16 patients) received a total dose of 70Gy to a planning target volume (PTVII) including also the base of the seminal vesicles, plus a boost of 6-8 Gy to the prostate only (PTVI). Group B  received a single course of treatment up to 78Gy to the entire PTV including prostate and base of seminal vesicles (or prostatic bed for patients who received surgery). In all cases, dose normalization was set to mean dose to PTV. In the framework of the initial phase of RapidArc clinical practice, no hypo-fractionation or dose escalation scheme was introduced and will be part of future investigations.Organs at risk routinely considered in these patients are rectum, bladder, femoral heads and penile bulb. Rectum was delineated from 1 cm above anus to the sigma tract. In addition, as practice for all intensity modulated patients, the Healthy Tissue (HT) was defined as the patient's volume included in the CT dataset minus the PTV volume. No specific immobilisation systems were applied to prostate patients as well as no strong requirements on patient preparation. In this respect, patients were asked to empty bladder about half an hour prior to treatment and to regularize rectal evacuation during the first two weeks of treatment, also using small glycerine based enema one hour before treatment. Routine institutional image-based patient position verification protocols foresee 2D-2 D matching of orthogonal kV-MV images acquired with the On Board Imaging system installed at the accelerator with evaluation performed by radiographers and application of couch shifts if total vector length of displacement is smaller than 7 mm. Cone Beam CT is becoming part of our routine protocol and is now performed once a week in addition to the 2D-2 D matching (kV-MV) most common procedure. The introduction of RapidArc and a more systematic application of image-based patient position verification did not lead, in this first phase of clinical practice to any modification in target or margin definitions which were kept, for this group of patient, the same as for the previously adopted 3 D conformal technique.95% > 98% and V107% = 0.0%. Concerning bladder the aim was to keep mean dose < 45Gy and D2% < 80Gy. Planning objectives for rectum were: mean < 45Gy, V50Gy < 50%, V60Gy < 40%, V70Gy < 15%. For femoral heads, dose objective was D2% < 47Gy. The dose of 30Gy was considered as objective for mean dose to penile bulb. No explicit planning objectives were set for healthy tissue.RA plans were optimised for single arcs  for a Clinac 2100iX equipped with a Millennium-120 MLC (120 leaves with a resolution at isocentre of 5 mm for the inner 20 cm and 10 mm for the outer 2 \u00d7 10 cm) and a photon beam energy of 6MV. Further details on RA technique can be found in ,5. Plan All dose distributions were computed with the Anisotropic Analytical Algorithm (AAA) implemented in the Eclipse planning system with a calculation grid resolution of 2.5 mm.Technical features of treatments have been reported in terms of main delivery parameters (field and control point (CP) size, MU, MU/deg and MU/Gy, Dose Rate (DR), Gantry Speed (GS), Collimator angle, beam-on time). Results of pre-treatment plan quality assurance are reported as Gamma Agreement Index (GAI), i.e. the percentage of modulated field area passing the \u03b3-index criteria of Low  with thr2%, D98%, V95%, V107%), homogeneity (D5%-D95%) and conformity (CI90%). CI90% is defined as the ratio between the volume of patient irradiated at 90% of the prescribed dose and the PTV volume. For OARs, the mean dose, the maximum dose (D2%) and appropriate values of VxGy (volume receiving at least \u00d7 Gy) were scored. For Healthy Tissue, the integral dose DoseInt was reported as well. This is measured as the integral of the dose delivered to the entire HT and is expressed in Gy cm3.Dosimetric quality of treatments was measured from the dose volume histogram (DVH) analysis. For PTV the following data were reported: PTV coverage . Toxicity scoring was assessed by non blind radiation oncologists in charge of the various patients and according to the National Cancer Institute Common Terminology Criteria of Adverse Effects scale (CTCAE version 3 ) as partFigure Table From the summary of main technical features it derives that treatment of prostate is characterised by relatively small field and control point areas resulting in a low output factor requiring high number of MU per minute and high average dose rate. With conventional fractionation and single arcs, gantry speed is kept constant at maximum speed.Pre-treatment quality assurances of RA plans resulted in an average gamma agreement index GAI 3% superior to the acceptance threshold of 95% set as reference in our institute.Dosimetric data showed that all planning objectives were met for PTVI and PTVII-PTVI (for group A only). Conformity of treatment, not explicitly considered as a planning objective, resulted acceptable. DVH analysis of organs at risk showed that all planning objectives were largely met when considering the fraction of organs not overlapping with PTV and when considering the entire organs (bladder and rectum) too.Clinical data summarized in table Based on the results of an intensive program of pre-clinical investigations performed at planning level -9 to assThe main objective of this first phase of clinical introduction of RA is the assessment of the possibility to administer to patients standard radiotherapy treatments and moreover to investigate the potentials of improvements. These results were easily achieved in this group of patients: rectum tolerance, derived from ,23 were The dosimetric results reported here might also support the activation of a second clinical phase, aiming to implement more aggressive fractionation schemes .Having achieved the aimed quality of treatments, investigations of technical features of delivered plans, in comparison with previously reported data for different groups of patients , allow sData reported in table Concerning clinical workflow, delivery of about 1700 RA fractions to prostate, confirmed the significant reduction of effective treatment time anticipated in the preclinical phase -9. For aThe smoother process of RA could decrease the duration of the treatment reducing the risk of intra-fractional internal organ motion. In fact, bladder or rectum deformation was reported by several investigations. As an example, ,27 usingIt is obvious that the present study cannot be considered as conclusive and that long term observation of patients is needed to measure outcome and late toxicity. These preliminary results are anyway encouraging further experience in this field.Forty-five patients with prostate carcinoma were treated with Volumetric Modulated Arc Therapy according to the RapidArc implementation in a clinical feasibility protocol. Quality of treatments resulted in an improvement of all planning objectives with regard to both target coverage and organs at risk sparing. Clinical outcome for early acute toxicity and assessment of biochemical outcome showed encouraging results. Future investigations will aim to appraise treatment of patients with inclusion of pelvic nodes and altered fractionation schemes. Long term outcome has to be evaluated with proper follow-up but the first phase achieved the primary goal to demonstrate safety and efficacy of RapidArc.LC acts as Scientific Advisor to Varian Medical Systems and is Head of Research and Technological Development to Oncology Institute of Southern Switzerland, IOSI, Bellinzona.No special competing interest exists for any other author.GP, LC and AF coordinated the entire study. Patient accrual and clinical data collection was done by GP, AR, ES and MV. Data analysis, physics data and treatment planning data collection was conducted by AC, GN, EV and AF. The manuscript was prepared by LC. All authors read and approved the final manuscript."}
+{"text": "STEPS is a stochastic reaction-diffusion simulation engine that implements a spatial extension of Gillespie's Stochastic Simulation Algorithm (SSA) in complex tetrahedral geometries. An extensive Python-based interface is provided to STEPS so that it can interact with the large number of scientific packages in Python. However, a gap existed between the interfaces of these packages and the STEPS user interface, where supporting toolkits could reduce the amount of scripting required for research projects. This paper introduces two new supporting toolkits that support geometry preparation and visualization for STEPS simulations. Advanced research on neuronal signaling pathways frequently requires assistance from computational modeling and simulations, causing the development of several molecular reaction-diffusion simulators in recent years. In this domain, the general assumption of mass action kinetics in a well-mixed volume is often invalid, whilst stochasticity and spatiality have been demonstrated to play essential roles in regulating behaviors of the system  is a dynamic programming language with many packages that are beneficial for scientific research, such as NumPy (http://www.numpy.org/) and SciPy (http://www.scipy.org/) for scientific computing, and Matplotlib (http://matplotlib.org/) for data plotting. The simplicity, readability and ultimate flexibility of the language have raised interest from the computational neuroscience community, where many simulators now support Python as their optional or even default user interfaces, including NEURON , a toolkit that integrates MCell with Blender (http://www.blender.org/), providing a complete solution for triangular surface mesh construction, component identification, MCell model association and simulation result visualization.Reaction-diffusion simulators commonly accept formatted text files as data input, where geometry is described either as a combination of predefined primitives like spheres and cubes, or as a surface or volume mesh. The data is then dealt with differently among simulators. SSA based simulators like MesoRD and NeuroRD generate cubic meshes according to the input primitive geometries, while particle based simulators like Smoldyn and MCell establish mathematical boundary representations of the geometries. Data files for simplified geometries can be produced manually, but the generation of complex or realistic geometries, like those based on reconstructions from electron microscopic imaging, often relies on third party professional applications. Therefore, toolkits that integrate the geometry generator and the simulator can be beneficial. One example is CellBlender (http://www.3ds.com/products-services/simulia/portfolio/abaqus/), TetGen (http://tetgen.berlios.de/), and Gmsh (http://geuz.org/gmsh/). To further enhance this interaction, we developed a Python-based toolkit that integrates STEPS with CUBIT (https://cubit.sandia.gov/), a sophisticated surface/volume mesh generator. CUBIT provides both commercial and academic licensing as well as a 30-day full trial version. There are several reasons that we choose CUBIT as the primary supporting application. Unlike MCell, which accepts triangular surface meshes as its geometry inputs and is thus able to utilize free surface mesh generators such as Blender, STEPS simulations require tetrahedral meshes that are not supported by those generators. Open source tetrahedral mesh generators such as TetGen and Gmsh remain focused on a non-interactive scripting based generation approach and are therefore unqualified for the mesh preparation tasks described here. CUBIT not only implements multiple tetrahedron mesh generation algorithms, from simple automatic approaches to complex, geometry adapting methods, but also embeds an interactive Python environment and a large set of Python base APIs, which enables flexible data and function integration with STEPS. It supports importing of multiple mesh formats including the Abaqus format, the primary mesh format used in STEPS. Additionally, CUBIT supports both primitive-based mesh generation that is suitable for simplified geometry generation, and a facet-based engine for realistic geometry reconstruction, and is therefore suitable for a wider range of research compared to other generators that support a single approach.Different from MesoRD and NeuroRD, STEPS does not generate meshes itself, but makes use of professional mesh generators. A generic mesh importing mechanism is provided, together with importing functions for common mesh formats such as Abaqus  dataset where element lists can be named and stored. ROI datasets are accessible by name once created. A set of ROI operation APIs are also implemented in STEPS so that stored elements can be reused in the simulation.To form a spatial reaction-diffusion system, groups of reaction and diffusion rules (\u201cvolume systems\u201d) defined in the biochemical model need to be added to corresponding compartments in the geometry, and groups of defined surface reaction rules and other surface phenomena (\u201csurface systems\u201d) need to be added to related patches. Volume systems and surface systems are defined separately in a steps.model.Model object. STEPS associates biochemical systems with geometry components by storing system ids in corresponding components in the geometry object. The model and geometry objects are then combined to construct the stochastic spatial solver (steps.solver.Tetexact). The separation of biochemical model definition and geometry description not only helps modelers to maintain focus, but also enhances the reusability of scripts as a single model definition can be reused with different geometries, and vice versa.In practice, biochemical model and geometry are often prepared by different individuals, therefore it is necessary for a Tetmesh to be stored in a file and retrieved later for simulation. This functionality is provided by the MeshIO utility, which saves and loads a Tetmesh object, compartment and patch definitions, biochemical model association and lists of element groups, to and from an xml file.Though geometry preparation can be accomplished manually using the above mechanisms, the toolkit combines these mechanisms and provides flexible pipeline functions in Python that significantly reduce the labor required. For example, selected tetrahedrons in CUBIT can be directly used to create a compartment with biochemical system association in Tetmesh geometry within a single function call in the toolkit, instead of going through the steps of index translation, compartment object creation and model association. This is particularly beneficial when using complex geometries.The importance of visualization for spatial reaction-diffusion simulations is a matter of debate. Though visualization provides an intuitive way for understanding simple biochemical models, its value for simulations with complex biochemical systems and geometries is unclear. This leads to divergent strategies in existing simulators. Some simulators, for example Smoldyn and MesoRD, implement built-in runtime visualization support. Other simulators such as MCell focus on post-simulation result playback using third-party applications. Both approaches have their advantages and disadvantages, thus whether a simulator supports one over another mainly depends on developer preference and application focus. Runtime visualization provides immediate information of how the simulated system behaves, important for model debugging and runtime simulation adjustment. However, a considerable amount of computational resource is required, reducing the overall efficiency of the simulator. Moreover, modern neuroscience simulations are often executed on clusters where no visualization is allowed. Therefore, runtime visualization is often implemented as an optional feature that can be switched off when necessary. Post-simulation result playback does not affect runtime performance of the simulation significantly, although history data storage is required. The amount of history data increases proportionally to simulation time, making this approach resource-consuming for long simulations. In addition, result playback can only be visualized after a simulation is completed, so it is unable to perform runtime adjustment of the simulation.STEPS implements a Python-based, interactive 3D visualization toolkit for spatial reaction diffusion simulations. Currently, the toolkit focuses on supporting runtime visualization, but simulation recording and playback will be added as extensions in the future. Despite the general understanding that visualization is limited to simulations with simple models and geometries and mostly for demonstration purposes, the STEPS visualization toolkit attempts to provide efficient, accurate and comprehensible visualization support for simulations with complex biochemical models and geometries, a goal that is not trivial to achieve. Here we detail the challenges encountered during the toolkit development and explain the solutions taken to tackle those challenges.The fundamental goal of the visualization toolkit is to visualize simulations with complex biochemical model and geometry. A major challenge lies in the presentation, that is, how to produce human comprehensible visual output of a complex system. Visualization support in existing simulators often adopt an \u201cAll-In-One\u201d strategy, where all molecules as well as the full geometry are displayed in a single window. Although this approach may be adequate for models with several reactions and simple geometries, due to the limitation of human perception, the visual output of such a presentation soon becomes incomprehensible as the complexity of the system increases.To address this problem, the STEPS visualization toolkit abandons the \u201cAll-In-One\u201d approach and introduces the \u201cComponent Assembly\u201d concept to the implementation instead. Figure Visual components are then assembled in a \u201cdisplay,\u201d an interactive 3D window environment that displays assigned components. One pivotal feature of the visualization toolkit is the \u201cMany-To-Many\u201d association between visual components and displays: instead of creating a single display window, the toolkit allows multiple displays to be created for a single simulation. Multiple visual components can be assembled in a display and each visual component can also appear in multiple displays. Visual components that appear in multiple displays maintain a single instance of internal data and synchronize their visual appearance among all displays when the data is updated during simulation, thus the increase of memory cost is insignificant.This implementation provides flexible solutions for different visual scenarios that may be encountered in practice. One common example is the \u201cGlobal-ROI\u201d scenario, where a single window displays all geometry components and molecule changes as a global view of system behavior, while a number of displays highlight changes of specific molecule species in different geometry regions. Another example is the \u201cSpecies of Interest\u201d scenario. In a complex simulation, molecules in different parts of the same geometry region often visually overlap with each other, significantly reducing the comprehensibility of the visualization. With the visualization toolkit, molecule species that are of interest can be isolated from the others and visualized in several displays separately, with the same static component as the geometry background of all displays.http://www.pyqtgraph.org/), a Python based scientific graphics and GUI library built on PyQt4 (http://www.riverbankcomputing.com/software/pyqt), PyOpenGL (http://pyopengl.sourceforge.net/) and NumPy. Visualization and interaction such as panning and rotation of views are handled directly by the package, allowing our implementation to focus on high level data representation instead of basic functionality coding. The package also supports runtime console interaction so that components can be added to or removed from displays to form new views of the simulation.Visual components and displays are extensions of generic OpenGL visual items provided by PyQtGraph  in Python, thus users can interact with the visual system freely even when the simulations are in execution in the background.Visualization of SSA-based spatial reaction diffusion simulation faces an intrinsic representation challenge that seldom appears in particle-based simulations, where the spatial position of each molecule is tracked and recorded accurately through simulation. The fact that SSA-based simulators do not track molecule movement but monitor the quantity changes of molecules in each subvolume means that the exact position of individual molecules is not known. Different approximations have been used to solve this problem. For instance, MesoRD allows users to predefine the maximum number of molecules that can be visualized per cubic subvolume. Based on this value, it then generates all possible molecule positions in advance by evenly partitioning the axes of the subvolume space. During simulation, each subvolume updates its condition iteratively and determines whether a molecule should appear on any of the positions. However, this approach was not suitable for STEPS visualization for several reasons. First, tetrahedral subvolumes have a much wider range of size and shape compared to the ones in a cubic mesh, thus it is practically difficult to partition the space evenly for each subvolume. Second, if all molecule positions are generated in advance it is possible for a molecule to be shown at a fixed position over time, giving the wrong impression that no movement has occurred for that molecule where instead conceptually it has changed position inside the subvolume. Third, as the maximum number of visible molecules is fixed for each subvolume, subvolumes with high concentrations of molecules may be visually over-simplified due to a lack of available positions, while the ones with low concentrations retain large amounts of unused coordinate data. Finally, the number of coordinates that need to be generated scales linearly with the number of subvolumes in the simulation, causing a large memory cost for simulations with fine meshes even if the amount of molecules in the system is small.Because of these reasons, the STEPS visualization toolkit, instead, adopts a runtime generation approach for molecule visualization. At each visual update iteration, tetrahedral and triangular SSA subsystems in every dynamic visual component calculate the number of molecules within themselves and generate the exact number of corresponding random positions. The toolkit uses a fast algorithm that guarantees all these random positions are uniformly distributed and bound by the subsystem's geometry. These positions are then fed to individual visual components and rendered in the corresponding displays as dots with different sizes and colors, predefined in the component. The process repeats when the simulation reaches the next visual update interval. One exception is the multiple-state \u201cchannel species\u201d on patches, whose positions are permanent after initialization except when they diffuse inside the membrane.3 for tetrahedron, and m2 for triangle). For each associated tetrahedron/triangle of the visual components, the maximum number of points that can be generated within is determined by multiplying the density with its volume or area, reflecting the proportional distribution of molecules. The density can be either predefined by the user, or adjusted automatically according to the ratio of maximum against actual amount of points that will be generated when the auto-adjust mode in the reducing function is enabled. Each visual component has its own maximum amount and density configuration so that they can be specified for individual species but remain consistent within the component.In the above solution, the number of random positions generated at each iteration equals the total number of molecules over all visual components. While this is achievable for simulations with a small numbers of molecules, as this number increases it becomes difficult and eventually unfeasible to render them due to limited graphical resources. Therefore the visualization toolkit regulates the position generation with two restrictions. The first restriction is the \u201cmaximum amount of points\u201d that can be generated for each visual component. Once the number of molecules in a component exceeds this maximum, a reducing function is called to lessen the amount of points generated according to the second restriction: \u201cmaximum point density,\u201d defined as the maximum number of possible points being generated per unit of measurement . Video recordings of these two examples are provided as Application of the above toolkits highly depends on the conditions and research interests of specific projects. In this section we present two examples that originate from our previous research to explain how the toolkits can be used in practice. The meshes and Python scripts used for these simulations can be downloaded from ModelDB (3R) model described in  and cytosol of a spine can be opened by first binding with cytosolic IP3 and then Ca2+, or can be inactivated by binding with Ca2+ directly. While open, IP3Rs release Ca2+ stored in the ER into the cytosol. Figure 3R state and the Ca2+ concentration increase in the cytosol.The first example is the inositol 1,4,5-trisphosphate receptor IPR model dhttp://synapses.clm.utexas.edu/anatomy/Ca1pyrmd/radiatum/K24/K24.stm) and artificially create a triangle mesh inside to represent the ER membrane of the spine (Figure 2+ bindings and transitions of different IP3 receptor sites are represented as surface reactions on the patch, and Ca2+ as well as IP3 are set to be diffusible in the cytosol compartment and Ca2+ is also diffusible in the ER compartment.To create a suitable geometry for the simulation, we extract a triangular spine morphology from an electron microscopic reconstruction of spiny dendrites (2+ in cytosol and ER is represented in orange, while IP3 in cytosol is represented in red, using the \u201ccompartment species\u201d visual component. Different IP3 receptor sites on the membrane are represented as different states of a \u201cpatch channel\u201d component with individual color and transparency configurations. Native and Ca2+ bound receptor states are colored in blue with different transparencies, while the IP3 bound state and the open state are colored in magenta with 20 and 100% opacity, respectively.In the visualization, Ca2+ ions and IP3 molecules present in the model. This is a common issue of visualization when dealing with complex simulations. Figure 3 receptor on the membrane can be clearly visualized in Figure 2+ concentration can also be seen in Figure Figure 3 receptor and cytosolic Ca2+ concentration, we create dynamic plots with these two measures and monitor their changes throughout the simulation. As shown in Figure 2+ activated an IP3 receptor at approximately 20 ms, leading to the release of Ca2+ from ER and the rapid increase of cytosolic Ca2+ concentration, which in turn increases the number of open-state IP3 receptors.In order to quantitatively analyze the relationship between the number of open states of the IPThe second example originates from our previous research (Santamaria et al., Four meshes were generated for this example, using project specified scripts for the CUBIT Python API. The mesh generation script is available upon request and can be modified to produce variations of the meshes. Each mesh consisted of a cylinder of 20 \u03bcm length and 0.7 \u03bcm diameter, representing the dendritic shaft. We then randomly attached a number of simplified spines, each formed by a spherical head and a cylindrical neck, onto the shaft cylinder. Spines were generated according to statistics from EM studies (Harris and Stevens, Four simulations are assigned to and executed by a simulation control, each of which simulates molecule diffusion in one of the four meshes. States of the simulations are visualized in separated displays. In each display, the mesh for the simulation is rendered by the compartment mesh component. As this research mainly focuses on the molecule distribution in the dendritic shaft, we use the shaft tetrahedron indices stored in the ROI dataset to create a tetrahedron species component that only displays molecules inside these tetrahedrons. This is a better solution compared to the one where all molecules in the simulation are displayed, particularly for meshes with high spine densities Figure . Visual To quantitatively visualize the difference of molecule distribution caused by varying spine density, we plot the spatial distributions along dendritic shafts using the visualization toolkit Figure . With in3R model example demonstrates how compartments and patches are identified and created in realistic spine morphology using the geometry preparation toolkit, and how the simulation can be visualized properly by splitting molecule species in multiple displays. The anomalous diffusion example showcases the usage of \u201cRegions of Interest\u201d datasets for visually filtering molecules in a specific region. This example also demonstrates how multiple simulations are executed and visualized simultaneously for comparison.In this paper we have described two supporting toolkits for STEPS that are implemented in Python. We've introduced the geometry preparation toolkit that integrates CUBIT with STEPS via Python, allowing complete mesh preparation solutions for STEPS simulations. We've also analyzed approaches to improve efficiency, accuracy and comprehensibility of visualization for spatial reaction diffusion simulations, which are adopted in our implementation of the visualization toolkit. Two examples are presented to showcase the application of the toolkits in real research projects. The IPThe toolkits are components of the STEPS supporting environment, where Python-based submodules are implemented to close the gaps between interfaces of various Python packages and the generic interface of STEPS. The Python world is an open and rapidly growing community where hundreds of new packages are available to the public everyday. On one hand, this provides rich and flexible package options for research projects using STEPS, on the other hand, packages selected to implement a customized toolkit may soon be out of date or lack improved features provided in new packages. Therefore, instead of detailing the package-dependent, technical implementation of the toolkits, we've concentrated on introducing novel, underlying mechanisms and principles involved. The approaches described in this paper are beneficial not only to the implementation of current toolkits, but also to the design and implementation of toolkits for other simulators in the same category.At the moment the STEPS supporting environment is not yet completed, and the existing toolkits can be further improved in several aspects. The generation of biochemical models remains text based, requiring significant amount of human efforts in scripting and maintenance, despite the availability of the SBML (Hucka et al., As for the toolkits described in this paper, the geometry support toolkit requires CUBIT, which is commercially licensed. We anticipate alternatives with similar functionality that can be obtained freely so that the whole geometry preparation process can be achieved without extra financial cost. One candidate is TetGen, whose format has been supported in STEPS since early versions, although it still lacks several features such as graphical interaction with meshes. So far, the visualization toolkit supports visualization of spatial reaction diffusion systems, but does not yet support visualization of new features in STEPS version 2, such as membrane potential and current, which is implemented in the EField system (Hepburn et al., http://steps.sourceforge.net.STEPS 2.2 with both toolkits described in this paper, as well as API references and a user manual, can be accessed from Weiliang Chen designed, implemented and tested the toolkits described, as well as drafted the manuscript. Erik De Schutter conceived of and supervised the STEPS project and helped draft the manuscript. Both authors contributed to the manuscript and read and approved the submission.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."}
+{"text": "NEB), which is thought to account for roughly 50% of cases.Nemaline myopathy (NM) is a congenital muscle disease associated with weakness and the presence of nemaline bodies (rods) in muscle fibers. Mutations in seven genes have been associated with NM, but the most commonly mutated gene is nebulin (NEB mutations: a point mutation in intron 13 and a frameshift mutation in exon 81. Levels of detectable nebulin protein were significantly lower than those in normal control muscle biopsies or those from patients with less severe NM due to deletion of NEB exon 55. Mechanical studies of skinned myofibers revealed marked impairment of force development, with an increase in tension cost.We describe two siblings with severe NM, arthrogryposis and neonatal death caused by two novel Our findings demonstrate that the mechanical phenotype of severe NM is the consequence of mutations that severely reduce nebulin protein levels and suggest that the level of nebulin expression may correlate with the severity of disease. TPM3) [ACTA1) [NEB) [TPM2) [TNNT1) [CFL2) [KBTBD13 [KBTBD13, whose function is unknown, these genes share the unifying feature that they all encode proteins of the sarcomeric thin filament, suggesting that weakness and rod formation in NM is related to improper thin-filament structure and function [With an estimated incidence of 1 in 50,000 live births, nemaline myopathy (NM) is the most common of the congenital myopathies, accounting for roughly one-half of the cases of these conditions [TPM3) , skeleta1) [NEB) , tropomy [TNNT1) , cofilin) [CFL2)  and KBTB[KBTBD13 . With thfunction .NEB gene is large, with a total of 183 exons spanning 249 kb of genomic sequence and a theoretical full-length transcript of 26 kb, and is predicted to encode an approximately 800-kDa protein [NEB exons, leading to production of at least hundreds of distinct isoforms [The skeletal muscle-specific  protein . Great disoforms ,12. A siisoforms . Controlisoforms -17. Nebuisoforms . Studiesisoforms ,21 and aisoforms .NEB gene are the most common cause of autosomal recessive NM [NEB mutations have also been reported in patients with \"intermediate\" and \"severe\" forms of NM, characterized by lack of ambulation or even death in infancy [NEB have been reported in NM probands [NEB exon 55, identified in Ashkenazi Jewish NM patients with variable forms of NM, has been studied extensively at the genetic and physiological levels and has been shown to result in moderately reduced levels of nebulin [NEB.Mutations of the skeletal muscle-specific ssive NM . Althougssive NM , NEB mut infancy . To dateprobands ,10,14. Lprobands . These s nebulin ,17. In tA North American family (family \"16\") with two affected siblings with severe NM was referred for research studies to determine the genetic basis for their condition.At the time of the birth of patient 16-2, the mother was 25-year-old G7, P2-2-2-4 . There was no family history of neuromuscular disease in either parent, and neither parent had signs or symptoms of muscle disease.This baby boy was born after a pregnancy complicated after 31 weeks by polyhydramnios, and fetal movements were weak and infrequent. Birth occurred at 37 weeks gestational age, and the boy required intubation in the delivery room. He was 47 cm in length, weighed 2,500 g, and had a head circumference of 37.5 cm. The patient had facial weakness; contractures of the hips, knees, ankles, elbows and wrists; and other abnormalities, including a broad, prominent forehead; downward-slanting palpebral fissures; micrognathia; a bulbous nose; a cleft palate; ears that were low-set and posteriorly rotated; cryptorchism; and a small phallus. His neurological findings were otherwise normal. He required tube feeding. Echocardiography revealed a large patent ductus arteriosus with pulmonary hypertension. Electromyography performed at one week of age was inconclusive. A biopsy of the right rectus femoris muscle obtained at eight days of life revealed myopathic muscle with numerous nemaline bodies and/or rods, which are diagnostic for NM (see below). He was ventilator-dependent until 28 days of life, when ventilator care was withdrawn and he was taken home. He died a few hours thereafter.In utero monitoring demonstrated poor fetal movement and contractures of the upper and lower extremities. At birth, contractures were present at the elbows, wrists, fingers, hips, knees and feet, with the first, second and fifth digits overlapping the third and fourth digits of the hands. The infant had significant respiratory distress at delivery, with no respiratory effort and poor color despite administration of 100% oxygen. Prior to delivery, the parents had requested supportive measures only, and active treatment was discontinued because of the patient's clinical findings and significant respiratory distress during his first day of life. The patient died shortly thereafter.This baby boy was born two years later to the same parents at 31 weeks gestational age by spontaneous vaginal delivery, with Apgar scores of 1, 1 and 2 at one, five and ten minutes, respectively. The pregnancy was complicated by oligohydramnios and preterm precipitous onset of labor. A right rectus femoris muscle biopsy taken at eight days of life revealed skeletal muscle with small, round fibers and excessive variation in fiber size Figure . NumerouThe causes of death reported on the basis of autopsy findings were severe congenital NM and patchy acute bronchopneumonia of the left lung. The lungs appeared mildly edematous but without discrete lesions on the cut surface. The heart and all other organs were reportedly unremarkable. The histological findings in cardiac muscle were normal at both light and ultrastructural levels.Frozen muscle from the psoas, quadriceps, diaphragm and cardiac muscles collected at the time of autopsy were available for histological review. Gomori trichrome staining of the psoas muscle Figure  revealedAt autopsy, pulmonary findings included bilobed right lung, markedly hypoplastic lungs bilaterally, and immature, minimally expanded lung parenchyma. Histologically, minimally expanded alveoli contained scattered squamous cells and possible early hyaline membranes. The right and left pulmonary veins were significantly smaller than expected (1 or 2 mm), but the relationship and size of the vasculature were otherwise normal. Sections taken from the thymus, trachea, esophagus, adrenal gland, spleen, kidney, pancreas, bone, bone marrow and brain were unremarkable when visualized by light microscopy. The heart was structurally normal for gestational age, with a patent ductus arteriosus and foramen ovale.Evaluation of skeletal muscle from the diaphragm Figure  revealedACTA1, TPM2, TPM3 and CFL2 genes, as well as the recurrent NEB exon 55 deletion, were all negative in one or the other of the two affected patients. Genomic PCR and denaturing high-performance liquid chromatography (dHPLC) analysis of 159 NEB gene (GenBank:NG_009382.1) exons revealed two distinct mutations, one each in the patients' father and mother. Both affected boys were compound heterozygotes for these two autosomal recessive mutations. The mutation inherited from the father was a splice site mutation in the 5' splice site of intron 13 (GT > TT)   Figure , and the) Figure . NeitherNEB showed labeling of nebulin's C terminus approximately 72% reduced in comparison to control [Western blot analysis performed using protein extracted from quadriceps muscle from patient 16-2 revealed significantly lower nebulin content in comparison to control patients and patients with exon 55 mutations who had been evaluated in a prior report . Protein control . Prior w control ,17 descrNEB exon 55 [2+-activated active tension (4 \u00b1 0.5 mN/mm2 compared with 88 \u00b1 5 mN/mm2 in control)  was dramatically decreased from 3.2 \u00b1 0.2 s-1 in control myofibers to 0.4 \u00b1 0.04 s-1, even more so than in the previously described patients [Recent studies using tissue from nebulin-knockout mice -22 and h exon 55 ,22 have ) Figure , which ieletions . Becausepatients  . Rather than a specific pathological diagnosis, AMC is a description of a clinical phenotype that occurs in 1 in 3,000 live births and is a characteristic of more than 300 different disorders . In caseNEB , but thiNEB, which encodes an N-terminal portion of nebulin [Neb-knockout mice [ktr and increases in tension cost. Whether the apparently greater diminution of ktr in muscle from patient 16-4 is really associated with these boys' unusually severe clinical presentations will require the identification and analysis of additional similar cases.A recent report described variably severe NM in patients with mutations in exon 55 of  nebulin . Mechani nebulin . Contracout mice , the patout mice ,22 and oNEB exons 3 and 22 [NEB.Our Western blot studies using antibodies against the N- and C-terminal portions of nebulin revealed significant reduction of nebulin content with greater immunoreactivity detected using antibodies against the C-terminal portion of nebulin rather than the N-terminal portion. Studies of patients with deletion of exon 55 have also detected reduced quantities of nebulin of appropriate molecular size and weaker immunoreactivity to antibodies directed against the N terminus . These f3 and 22 . In thatNEB mutations can cause marked impairment of contractile performance that causes severe myopathic disease. The compound heterozygous mutations described in this report represent another scenario in which NEB mutations can cause severe NM that is associated with an even shorter life expectancy than that of the most severely affected patients with exon 55 mutations. Single-fiber contractile studies from patient 16-2 in this study revealed marked decreases in contractile force compared with control fibers, and these decreases exceeded the contractile force deficits reported in studies using myofibers from NM patients with mutations in exon 55 of NEB [NEB. Additionally, our results imply that low levels of nebulin protein detected by Western blot analysis may correlate with a poor prognosis for patients with NM due to NEB mutations, which would be diagnostically useful if this finding holds true in studies of larger numbers of patients.Our studies provide further evidence that 5 of NEB . OverallMuscle biopsy and autopsy tissues were obtained and prepared using standard histological protocols . The recNEB gene (GenBank:NG_009382-1) was performed by dHPLC and sequencing as previously described [Mutation analysis of the escribed . The dHPBefore dHPLC analysis, PCR samples were denatured for 3 minutes at 95\u00b0C and then slowly reannealed by lowering the temperature from 95\u00b0C to 37\u00b0C over a period of 1 hour. Two to five microliters of the PCR amplicon on the 96-well plate were injected into a heated reverse-phase DNASep Column . The column temperature of the dHPLC was set for partially denaturing conditions. The melting profiles of the amplicons were calculated using the Navigator software, but the exact temperature was determined empirically. Conditions used for dHPLC analysis of each amplicon are available on request (from VLL).Following dHPLC analysis, samples showing abnormal peaks were sequenced. The PCR products were purified using Exonuclease I and shrimp alkaline phosphatase , and the purified products were sequenced using BigDye version 3.1 sequencing chemistry and an ABI 3730 DNA Analyzer . Sequences were analyzed using Sequencher 4.1 software .For determination of nebulin content, muscle samples  were first homogenized in buffers containing protease inhibitors  to prevent protein degradation during the homogenization process. The homogenized muscle samples were run on 2.6% to 7% SDS-PAGE gels, and transferred onto polyvinylidene fluoride membrane using a semidry transfer unit . Blots were stained with Ponceau S to visualize total transferred protein. The blots were then probed with primary antibodies against nebulin's N terminus  and its C terminus  ,32 or ag+-ATP, 1 mmol dithiothreitol, 46.35 mmol K+-propionate, 15 mmol creatine phosphate, pH 7.0, at 20\u00b0C) containing 1% (vol/vol) Triton X-100. Control samples for muscle mechanics studies were isolated from the quadriceps muscles of three living individuals between 30 and 40 years of age, and all results were comparable to previously published results for control and experimental specimens representing a variety of ages, muscle groups and postmortem or postbiopsy intervals [2+. Preparations were washed thoroughly with relaxing solution and stored in 50% glycerol relaxing solution at -20\u00b0C for up to approximately 8 weeks. Small muscle bundles (diameter approximately 0.07 mm) were dissected from the skinned strips and mounted between a displacement generator and a force transducer element  using aluminum T clips. Sarcomere length (SL) was set using a He-Ne laser diffraction system. Mechanical experiments performed on contracting muscle were carried out at a SL of about 2.5 \u03bcm for control muscle and at just over slack length for NM muscle, a length selected on the basis of our prior studies. By constructing force-SL relationships, we previously showed that at a SL of 2.5 \u03bcm, human muscle fibers from controls produced maximal force, whereas nebulin-deficient muscle fibers from NM patients produced maximal force just over slack length because of their shorter thin filaments [Small strips dissected from muscle biopsies were skinned overnight at about 4\u00b0C in relaxing solution -2-aminoethane sulfonic acid (BES), 10 mmol ethylene glycol tetraacetic acid (EGTA), 6.56 mmol MgCl2, 5.88 mmol Nantervals ,22. To eilaments . Thus, bilaments .et al. to measure simultaneous force-ATPase activity [-1 pyruvate kinase (500 U mg-1), 0.24 mg mL-1 lactate dehydrogenase (870 U mg-1) and 20 \u03bcmol diadenosine 5'-pentaphosphate. For efficient mixing, the solution in the bath was continuously stirred by means of motor-driven vibration of a membrane positioned at the base of the bath. ATPase activity of the skinned fiber bundles was measured as follows. ATP regeneration from adenosine diphosphate (ADP) was coupled to the breakdown of phosphoenol pyruvate to pyruvate and ATP was catalyzed by pyruvate kinase, which is linked to the synthesis of lactate catalyzed by lactate dehydrogenase. The breakdown of NADH, which is proportional to the amount of ATP consumed, was measured online by UV absorbance at 340 nm. The ratio of light intensity at 340 nm (sensitive to NADH concentration) and the light intensity at 410 nm  was obtained by means of an analog divider. After each recording, the UV absorbance signal of NADH was calibrated by multiple rapid injections of 0.25 nmol of ADP (0.025 \u03bcL of 10 mmol ADP) into the bathing solution with a stepper motor-controlled injector. The slope of the ATP concentration versus time trace during steady-state tension development of a calcium-induced contraction , where F is force at time t and ktr is the rate constant of tension redevelopment.To measure approach  to disendHPLC: denaturing high-performance liquid chromatography; MHC: myosin heavy chain; NM: nemaline myopathy; PCR: polymerase chain reaction.The authors declare that they have no competing interests.MWL interpreted the clinical information, performed the pathological analysis, and prepared the manuscript. CAO and HG performed the contractile studies and composed the section of the results and discussion pertaining to contractile performance. VLL, KP, and CWP carried out the molecular genetics analyses, assisted in analyzing the clinical and genetic data and composed sections of the introduction and discussion related to these topics. KC performed genetic analysis on the patients and created the figure related to these data. AHB conceived of the study, participated in its design and data interpretation, and helped to draft the manuscript. All authors read and approved the final manuscript."}
+{"text": "AbstractHere we present a complete list of all valid species-group taxa of freshwater gastropods reported from Miocene and Pliocene deposits in Europe. The last comparable work dates back to the 1920s and covered about 1,600 names. The extensive literature research underlying the present work revealed considerable changes in the taxonomic and systematic frameworks of Neogene freshwater gastropods and yielded a total number of 2,156 accepted taxa. Each taxon is accompanied by a full citation of its first description; where the information is available, page number and illustration reference are provided. First descriptions available as open-access full-text sources on the web were linked via hyperlink to the first page of the publication. PageBreak1923. For the freshwater Neogene . We checked for correct spelling and nomenclatural validity (to exclude nomina nuda). Where feasible, we tried to include the full citation with indication of page number and illustrations (if present). Despite much effort it was not possible to acquire all of the mentioned publications. In such cases reference and indication of pages/illustrations were taken from PageBreakmost recent and accurate age attributions for the localities containing the relevant taxa, there are still doubtful cases where species may prove not to derive from Neogene sediments. Erroneous records of extant species in Neogene deposits were not considered herein.The list contains 2,156 accepted species-group taxa recorded for the Miocene and Pliocene. Since stratigraphical boundaries changed in the past decades and the age attributions of many gastropod-bearing localities have been revised since then, many taxa originally recorded for \"Miocene\" or \"Pliocene\" localities have been shown to belong to earlier or younger strata and vice versa. Although we tried to find out the Melanopsis atanasiui, Caspia dacica, and Prososthenia pertica. The publications mentioned in the species list Source hyperlink: Hyperlink to first page of reference, if availableAlthough much time was spent to acquire all the relevant literature, this list may be incomplete. Moreover, not every described species is incorporated here, since the list only includes accepted names. Unaccepted names, such as primary homonyms, junior synonyms, nomina nuda, nomina dubia, or nomina inquirenda are not covered. For those older than 1923 see Where a species contained one or several subspecific taxa (or they are currently ranked as such), the nominal subspecies was excluded from the list, so as not to overstate the actual number of accepted names."}
+{"text": "Typical RF levels in the classroom were similar between Wi-Fi and radio but higher than other sources. In the schoolyard typical RF levels were higher for radio, TV and mobile phone base stations compared to Wi-Fi. The results of this study showed that the typical RF exposure of children from Wi-Fi at school is very low and comparable or lower to other sources in the environment.The increasing use of Wi-Fi in schools and other places has given rise to public concern that the radiofrequency (RF) electromagnetic fields from Wi-Fi have the potential to adversely affect children. The current study measured typical and peak RF levels from Wi-Fi and other sources in 23 schools in Australia. All of the RF measurements were much lower than the reference levels recommended by international guidelines for protection against established health effects. The typical and peak RF levels from Wi-Fi in locations occupied by children in the classroom were of the order of 10 The use of Wi-Fi technology has become increasingly common in many places throughout the community, including schools. Through the use of this technology, electronic devices are connected to a computer network wirelessly using radiofrequency (RF) electromagnetic fields, thereby eliminating or reducing the need for network cables in the classrooms and other places. A common example is a laptop or tablet connected to the internet via Wi-Fi access points installed around the school..Wi-Fi is a type of wireless local area network which operates in unlicensed regions of the RF spectrum in the 2.45 and 5 GHz bands. The technology is designed to be used up to a few tens of metres between a device and an access point. Over these short distances Wi-Fi devices only use low output power, typically limited to 2 W or less. Children in a Wi-Fi enabled school are exposed to low level RF fields intermittently when using devices on the network and also from the access points and some portion of the transmitted RF energy is absorbed within their bodies has given rise to public concern about the RF exposure from Wi-Fi equipment, particularly in schools. Some individuals and groups including parents have publicly expressed concern that RF exposure from the technology has the potential to adversely affect children as well as the general population. Moreover, there are groups of concerned citizens actively campaigning against the installation and use of wireless technologies in schools and other public places.The increasing popularity of Wi-Fi technology, 5. The guidelines developed by the International Commission on Non-Ionizing Radiation Protection (ICNIRP) in particular form the basis for regulations within most parts of the European Union and many other countries including Australia. The exposure limits in the ICNIRP guidelines, which include basic restrictions and indicative reference levels for measurement, are intended to protect people of all ages and health status against all established adverse health effects that result from excessive RF exposure.International exposure guidelines for RF fields have been developed on the basis of current scientific knowledge to ensure that RF exposure is not harmful to human health\u20139. Although Wi-Fi clearly operates at low power, little data is currently available on typical RF exposures from wireless networks in schools, 10. It is therefore important to measure the RF exposure of children from Wi-Fi in schools and compare it with the ICNIRP exposure guidelines, but also with exposures from other common sources of RF in the environment.A limited number of previous measurement surveys have shown that exposure to RF fields from Wi-Fi in public places is expected to be much lower than the reference levels for public exposure specified in the ICNIRP guidelinesIn the present study the Australian Radiation Protection and Nuclear Safety Agency (ARPANSA) conducted measurements of RF electromagnetic fields from Wi-Fi and other sources in 23 schools located in two states in Australia. The main aims of the study were to measure the typical and peak RF exposure from Wi-Fi in the classroom and schoolyard and compare these against the public exposure reference levels of the ICNIRP guidelines. In order to better understand the RF exposure environment in these schools, the Wi-Fi measurement results were also compared to RF exposure from other sources in the everyday environment, such as mobile phone base stations, radio and TV towers and other sources.Schools were selected from the two most populated states in Australia, New South Wales (NSW) and Victoria. ARPANSA initially engaged with the education departments in Victoria and NSW in order to seek permission to conduct the study and acquire a list of possible schools that can be invited to participate in each state. The Victorian Department of Education and Training provided a list of 220 schools in Victoria with multiple Wi-Fi access points. The list of Victorian schools was classified into 10 groups consisting of a mixture of metro and rural schools, secondary and primary schools, smaller and larger schools (according to student numbers), and schools with a small and large number of access points. From each of the 10 groups one school was randomly selected to be invited to participate. The NSW Department of Education provided a list of 17 schools that self-nominated based on a bulletin about the study that was circulated to all NSW schools by the Department.The 10 Victorian and 17 NSW schools were invited to participate in the study during June 2016. The letter of invitation included an information pack explaining the reasons for the study and a summary of the measurement protocol. Initially three Victorian and 10 NSW schools agreed to participate in the study. A further 53 Victorian schools were selected and invited to participate during June-August 2016; of these, nine agreed to participate. The NSW Department of Education in July 2016 requested that another school which had experienced parental concerns regarding Wi-Fi at the school be included in the study. In total 23 schools, 12 in Victoria and 11 in NSW participated in the study. Different characteristics of the schools are shown in Table The participating schools were visited for measurements during June to September 2016. All measurements were performed via appointment mainly during school hours between 8.30 am and 3.30 pm; one school was measured during school holidays and another during the school's sports day where all the students were off campus. All the measurements were performed by technically trained ARPANSA staff members.2).RF fields were measured using a calibrated Narda SRM\u20133006 Selective Radiation Meter  and three separate tri-axial probes (one magnetic and two electric field probes) covering different frequency ranges from 9 kHz to 6 GHz. The meter was set to record the power flux density of the RF field  the measurements were conducted in an empty classroom to avoid lesson disruption; in two schools the classroom was measured with students present at the request of the principal and in another the classroom was measured with a group of teachers present that were preparing a lesson plan.The measurements included recording the average and maximum RF fields due to Wi-Fi whilst moving throughout the classroom; detailed measurements of the specific frequency bands used by Wi-Fi technologies at several stationary positions within the classroom; and recording all detectable RF signals up to 6 GHz, representing different RF sources, in the classroom and in the schoolyard.Spatial measurements of RF fields from Wi-Fi were recorded whilst walking slowly throughout the classroom and sweeping the probe slowly up and down (up to head height) over a period of 10 min (idle mode). All readily accessible locations within the classroom were visited at least once, paying particular attention to student desks and locations close to the nearest Wi-Fi access point. This procedure was repeated whilst downloading (or uploading) large files, browsing the internet or otherwise interacting with the Wi-Fi using one or more laptops in the classroom (active mode). During both the idle and active mode walk-throughs the average and maximum RF fields over the ten-minute period were recorded representing the typical and peak exposure in locations usually occupied by students in the classroom.Nominal centre of the classroom.Nearest student desk to access point.Furthest student desk to access point.At the access point, either directly underneath a ceiling mounted access point or 0.5 m from the wall of a wall mounted access point.Measurements of RF fields from Wi-Fi were conducted at 1.5 m above the ground (representing the head/torso of a child), with the probes mounted on a tripod, at stationary locations in the classroom for one minute whilst the Wi-Fi was active. The stationary locations included:The average and maximum RF fields from Wi-Fi over the 1-min period for each location were recorded.At the nominal centre of the classroom, RF fields were measured at 1.5 m above the ground for 1 min in various frequency bands across the spectrum representing different RF sources including (AM and FM) radio, TV, mobile telephone base stations (downlink only), Wi-Fi and other sources; these are listed in Table 2; for Wi-Fi the ICNIRP reference level is 10 W/m2.The RF levels that were measured are presented both as power flux density values and as percentages of the power flux density reference levels for the general public recommended in the ICNIRP Guidelines. Depending on the frequency of the RF source, the ICNIRP reference levels vary from 2 to 10 W/mDescriptive statistics were calculated for all the measurements. Measurement distributions were tested for normality using the Shapiro\u2013Wilk test. Comparisons between idle and active Wi-Fi measurements were tested for statistical significance using the Wilcoxon signed-rank test. Comparisons between measurements of Wi-Fi at different locations in the classroom and Wi-Fi compared to other RF sources in the classroom and schoolyard were tested for statistical significance using the Mann\u2013Whitney test.\u03b1-level of 0.05.The effect of different school characteristics  on RF levels from Wi-Fi were investigated using multivariate linear regression. All the analyses were performed with the SPSS software  and the level of significance was set at an p < 0.01 for all) so they are better described by nonparametric statistics.All the RF levels measured in the 23 schools were much lower than the exposure reference levels of the ICNIRP Guidelines. The measurements showed a lognormal distribution (\u22124% ICNIRP reference level) compared to when it was idle (5 \u00d7 10\u22124%); significance of difference between the idle and active walkthrough averages was p < 0.01. There was no statistically significant difference between the walkthrough maximum in all the schools (peak exposure) when the Wi-Fi in the classroom was active compared to idle (p = 0.12).The RF levels for the walkthrough Wi-Fi measurements taken in the classrooms of the 23 schools under the idle and active conditions are shown in Figure \u22124 and 10\u22122% of the ICNIRP reference level, respectively at the furthest desk compared to 4 \u00d7 10\u22124 and 4 \u00d7 10\u22122% at the closest desk . The RF levels were similar between measurements conducted next to/under the access point (1.3 m) and the nearest desk to the access point .The RF levels for the stationary Wi-Fi measurements taken at different locations in the classroom are shown in Figure p = 0.46) but higher compared to other sources . The peak RF levels in the classroom were higher for Wi-Fi compared to all other sources, including radio  and also higher than all other sources combined (p < 0.01).The RF levels for the measurements of all RF sources taken in the centre of the classroom and in the schoolyard are shown in Figures p < 0.01 for all). Similarly the peak RF levels in the schoolyard were higher due to radio and mobile phone base stations compared to Wi-Fi (p < 0.02 for both) but Wi-Fi was higher than TV (p < 0.01).In the schoolyard the typical RF levels due to radio, TV and mobile phone base stations were higher compared to Wi-Fi (p = 0.98) but the peak exposure was higher in the classroom compared to the schoolyard (p < 0.01), mainly due to the maximum Wi-Fi signal.For the total RF levels from all sources the typical exposure was similar between the classroom and schoolyard .The multivariate linear regression showed that none of the school characteristics had an effect on the measured RF levels from Wi-Fi  in a classroom; stationary measurements of Wi-Fi at different locations in the classroom and measurements of all RF sources in the classroom and the schoolyard.. The average  and maximum (peak) RF levels from Wi-Fi in locations occupied by students in the classroom were of the order of 10\u22124 and 10\u22122% of the ICNIRP reference level, respectively. This was expected given the low output power of Wi-Fi equipment and measurement data from previous studies. Typical exposure to Wi-Fi in public places was also found to be well within the ICNIRP guidelines by Schmid et al., Foster and more recently by Industry Canada. Specifically investigating schools, Peyman and colleagues, 10 found maximum RF levels due to Wi-Fi in the order of 10\u22122% of the ICNIRP reference levels at distances of 1\u20132 m from the access point. More recently, Gledhill measured RF levels in different classroom locations of two schools and found average and maximum levels of less than 10\u22122% and 3 \u00d7 10\u22122% of the ICNIRP reference levels, respectively.All the RF levels measured in this study were much lower than the exposure reference levels for the general public recommended by the ICNIRP guidelinesIn the current study the 10-min walkthrough survey in the classroom showed that the typical RF levels were only slightly higher when the Wi-Fi in the classroom using one or more laptops was active compared to when it was idle. However there was no difference in the peak RF levels when the Wi-Fi in the classroom was active compared to idle. The access point regularly transmits short duration beacon signals to enable client devices (e.g. laptops) to identify and synchronise with the network. These beacon signals are transmitted at full power even when no device is connected (idle mode). Additional bursts are transmitted from the access point when communicating with a connected device (active mode). In the active mode the access point is sending signals more often but not at any higher power than in idle mode. Hence, there is no difference in the maximum detected signal between active and idle mode, but the average measured signal is higher in the active mode compared to the idle mode.et al. and Gledhill. Interestingly, in our study there was no significant difference in the RF levels measured between next to/under the access point and the nearest desk to the access point. Although the positioning of the access point in the classroom of the 23 schools varied quite substantially it was often quite close to the nearest desk.For the stationary Wi-Fi measurements conducted in the classroom the current study showed that the RF levels were slightly higher in the nearest desk to the access point compared to the furthest desk. This was expected since RF decreases with the inverse-square of the distance and assuming that there is no other access points present in close distance. A similar pattern of decreasing RF levels with increasing distance from the access point was shown by Peyman \u22124% ICNIRP reference level). The peak exposure was higher for Wi-Fi compared to other sources and this was due to the beacon signal from the Wi-Fi transmitting at full power as explained earlier. The measurements conducted in the schoolyard showed that the typical RF levels from other sources such as radio, TV and mobile phone base stations were higher compared to Wi-Fi. Access points in schools are mainly installed for indoor coverage. A previous survey conducted by ARPANSA measured RF levels from different sources at 41 outdoor locations across Melbourne, Australia. This previous study also showed that the RF levels from broadcast antennas and mobile phone base stations were higher compared to Wi-Fi. Similarly Joseph et al., 14 showed that average RF levels measured in five European countries from various sources in different urban settings  were generally higher from broadcast and mobile telephony transmissions compared to Wi-Fi.Comparing Wi-Fi to other RF sources, the current study showed that the typical RF levels in the classroom were similar between Wi-Fi and radio (in the order of 10The current study showed that none of the school characteristics had an effect on the measured RF levels from Wi-Fi. It was expected that the type (primary/secondary) and location  of the school would not have an influence on the results. There was a linear correlation between number of students and number of access points which was expected given that a larger school would require more access points to service its campus. This study showed that having more students and more access points does not have a major influence on the personal exposure of each student to Wi-Fi which will be largely dominated by the closest access point or client device rather than the total number of access points around the school.. The proportion of time that Wi-Fi transmits RF signals is called the duty cycle. Joseph et al. in measuring Wi-Fi in 176 different urban locations  found a median duty cycle of 1.4% over all the measurements. Particularly in schools, Khalid et al. in measuring Wi-Fi in six schools found a mean duty cycle from the access points of 4.8%. In our study duty cycle was measured separately for the 2.45 and 5 GHz transmissions when performing the stationary Wi-Fi measurements in the centre of the classroom. The median duty cycle for 23 schools that were measured in the current study was 6.3 and 2.4% for 2.45 and 5 GHz transmissions, respectively.Wi-Fi transmissions consist of sequences of RF burst signals or pulses ranging in duration depending on the amount of data being carried by a pulse. For the majority of schools (20) the measurements in the current study were conducted in an empty classroom (to avoid lesson disruption) with an access point and one laptop. In three schools, measurements were conducted with students or teachers present and using Wi-Fi devices. A comparison between measurements conducted in empty classrooms and classrooms with multiple students/teachers using Wi-Fi showed no significant difference in the RF levels ; although this may have been due to low numbers (only three schools measured with multiple users in the classroom).Members of the public often ask about the cumulative exposure that a child receives when using a Wi-Fi device in a classroom in which a number of children are simultaneously using Wi-Fi. When downloading files, most of the transmissions will be from the access point, not the students\u2019 device. When downloading and uploading only a portion of the maximum capacity of a network would be used even in a classroom filled with children using Wi-Fi. The Wi-Fi network divides RF transmissions among the access points and client devices therefore the individual RF exposure to a child in a classroom that is using a device consists of sequential exposures from all active devices, the majority of which are located at some distance awayThe results of this study showed that children's exposure to RF fields from Wi-Fi in schools is several orders of magnitude below exposure reference levels recommended by international guidelines for protection against established health effects. Further, the exposure from Wi-Fi is typically comparable or lower to other common sources in the environment."}
+{"text": "SEER). Frequency and rate analyses on demographics, stage, and survival were compared among non\u2010Hispanic whites, Hispanics, African American, and Asian/Pacific Islanders. A total of 18,124 cases were reported in SEER from 1973 to 2009 comprising 1.4% of all reported gastrointestinal cancers. Gallbladder cancer was more common in females than males . The age\u2010adjusted incidence rate was 1.4 per 100,000, significantly higher in females than males (1.7 vs. 1.0). Trend analysis showed that the incidence rate has been decreasing over the last three decades for males. However, among females, the incidence rate had decreased from 1973 to mid\u201090s but has remained stable since then. Trend analysis for stage at diagnosis showed that the proportion of late\u2010stage cases has been increasing significantly since 2001 after a decreasing pattern since 1973. Survival has improved considerably over time, and survival is better in females than males and in Asian/Pacific Islanders than other racial groups. The highest survival was in patients who received both surgery and radiation. Trend analysis revealed a recent increase of the incidence of late\u2010stage gallbladder cancer. Highest survival was associated with receiving both surgery and radiation.Primary gallbladder cancer is an aggressive and uncommon cancer with poor outcomes. Our study examines epidemiology, trend, and survival of gallbladder cancer in the United States from 1973 to 2009. We utilized the Surveillance Epidemiology and End Results database ( Primary gallbladder cancer (GBC) is a rare gastrointestinal malignancy but is the most common cancer arising in the biliary tract, representing 80\u201395% of all biliary tract cancers worldwide Most of the GBC cases are found incidentally in patients undergoing either laparoscopic or open exploration for cholelithiasis and/or cholecystitis. It is estimated that GBC can be found in 2% of cholecystectomies Gallbladder cancer has been understudied. Only few studies have been published in the last decade on epidemiology and survival of GBC in the United States SEER, a program of National Cancer Institute (NCI) is a source of population\u2010based cancer surveillance information in the US. SEER collects information on incidence, prevalence, and survival from specific geographic areas representing 28% of the total US population in 2010. The SEER Cancer Incidence Research Database consists of tumors reported to 18 registries since 2000, 13 registries since 1992, and nine registries since 1973. Geographic areas were selected for inclusion in the SEER program based on their ability to operate and maintain a high\u2010quality population\u2010based cancer reporting system and for their epidemiologically significant population subgroups. The population covered by SEER is comparable to the general US population with regard to measures of poverty and education. The SEER population tends to have a higher proportion of foreign\u2010born persons than the general US population From 1973, SEER used the following broad racial categories: white, African American, American Indian/Alaska Native, Asian/Pacific Islander, and other. Hispanics were identified from the NAACCR Hispanic Identification Algorithm (NHIA) (Hispanics and non\u2010Hispanics) variable. Our study population included patients diagnosed with GBC residing in SEER registries areas and reported in SEER database from 1973 to 2009 We used SEER*Stat and SAS software for the descriptive analyses SAS was used to compare the distribution of GBC cases by descriptive variables across race/ethnicity groups P value of 0.05 or less was considered statistically significant.The proportional hazard model's main independent variables included whether the patient had surgery or radiation. Additional covariates included age in 5\u2010year groups, year of diagnosis in 5\u2010year periods, race in four groups, marital status, sex, histology in nine groups, stage and grade. Missing values were assumed to be missing at random and those cases were simply dropped from the analysis. A two\u2010tailed P\u00a0<\u00a00.0001) more common in females (71%) than males (29%). Most of the cases were reported in NHW (66%) followed by Hispanics (16%), while 9% of cases were reported in AA and 8% cases in A/PI. Compared to NHW (71%), more Hispanics females (78%) and less A/PI females (63%) were diagnosed with GBC (P\u00a0<\u00a00.0001).A total of 18,124 incident cases of GBC were reported in SEER database from 1973 to 2009. It comprises about 1.4% of all gastrointestinal malignancies reported in the database during this time period. Table\u00a0P\u00a0<\u00a00.0001). In the SEER\u201013 from 1992 to 2009, the rates are significantly higher for AA, A/PI, and Hispanics than NHW for both males and females. Moreover, the rate for females is higher than males across each of these four race/ethnic groups. Trend analysis showed that the incidence rates for both males and females have generally decreased significantly since 1973  were selected for inclusion in survival analysis. Only patients who were diagnosed with GBC as their first reportable malignant primary tumor were included in survival analysis. Table\u00a0Gallbladder cancer is an uncommon malignancy of the hepatobiliary tract. Currently GBC ranks fifth among gastrointestinal cancers. The global rates for GBC exhibit significant variability, reaching epidemic levels for some specific geographic regions and ethnicities. The basis for this wide variance most likely resides in differences in environmental exposures, incidence, and prevalence of risk factors for GBC During the study period, incidence rate of GBC remained low in comparison to other gastrointestinal or hepatobiliary malignancies. The global historic data also suggest that it remained low in many other parts of the world Our study shows that survival of GBC is improving significantly over time. Survival is better in females than males and in A/PI than other groups. Patients who did not receive any surgery had higher RHs for GBC\u2010specific and any cause of death in comparison to patients who received surgery as either the only modality of treatment or in combination with radiation. Among patients who did not receive radiation, not receiving surgery was also associated with a higher hazard of a non\u2010GBC\u2010specific cause of death. Similarly, not receiving radiation was associated with higher relative hazards for all three end\u2010points. The highest survival was in patients who received both types of treatment. The higher hazards for non\u2010GBC\u2010related death associated with no treatment suggests that patients who did not receive treatment were at a higher risk of death overall. The higher survival in patients who underwent treatment may reflect in part the higher expectation of survival in those patients compared to those who did not undergo treatment. The information provided by SEER, however, remains limited and does not allow for further assessment of various treatment modalities on survival. Randomized controlled trials are needed to further evaluate the effects of surgery and/or radiation on survival. Nevertheless, this study provides valuable contribution to the literature by conducting a powerful analysis of the variables impacting survival, including the observed beneficial effect of surgery and radiation, in large number of cases analyzed over approximately four decades. The beneficial survival impact of surgical approach on gallbladder cancer has been mixed. However, an aggressive surgical approach for patients with gallbladder cancer has been reported to substantially and significantly improve survival of gallbladder cancer patients In this epidemiological study, we used the comprehensive and quality\u2010controlled SEER database, which adds significant strength to our analysis. The SEER\u201018 registries which have reportable tumors from the year 2000 and later covered more than 28% of the total US population in 2010; cases linked to population data with the major race/ethnic categories are available back to 1992 for 13 of the registries; and data on tumors diagnosed back to 1973 are available for the SEER\u20109 registries with the major race/ethnic categories but with population race data only in the groups categorized as white, AA, and other. The population covered by SEER registries program is highly comparable to the general US population; however, it tends to be somewhat more urban than rural and has a higher proportion of foreign\u2010born persons than the general US population In conclusion, GBC is rare cancer of the gastrointestinal tract, but its incidence has generally decreased over the past several decades and appears to have stabilized for females. We also document a trend change in the staging pattern of GBC revealing the proportion of cases diagnosed at distant stage increasing in recent years after a long period of decline since the 1970s. Along with these findings, we have revealed a significantly improved survival of GBC over time. Highest survival was associated with receiving both surgery and radiation.None declared."}
+{"text": "The following information is missing from the Funding section: This study was also funded by a grant from the Collaborative Research Center / Transregio 124 consortium \u201cPathogenic fungi and their human host: Networks of Interaction\u201d  to AAB, ES, KR, MM, IDJ and KV."}
+{"text": "Fluorescence microscopy is one of the workhorses of biomedical research and laboratory diagnosis; however, their cost, size, maintenance, and fragility has prevented their adoption in developing countries or low-resource settings. Although significant advances have decreased their size, cost and accessibility, their designs and assembly remain rather complex. Here, inspired on the simple mechanism from a nut and a bolt, we report the construction of a portable fluorescence microscope that operates in bright-field mode and in three fluorescence channels: UV, green, and red. It is assembled in under 10 min from only six 3D printed parts, basic electronic components, a microcomputer (Raspberry Pi) and a camera, all of which can be readily purchased in most locations or online for US $122. The microcomputer was programmed in Python language to capture time-lapse images and videos. Resolution and illumination conditions of the microscope were characterized, and its performance was compared with a high-end fluorescence microscope in bright-field and fluorescence mode. We demonstrate that our miniature microscope can resolve and track single cells in both modes. The instructions on how to assemble the microscope are shown in a video, and the software to control it and the design files of the 3D-printed parts are freely available online. Our portable microscope is ideal in applications where space is at a premium, such as lab-on-a-chips or space missions, and can find applications in basic and clinical research, diagnostics, telemedicine and in educational settings. Fluorescence microscopy is an essential tool in biomedical research used to visualize, analyze and study molecules, cells and tissues. One of its main applications is to enable the quantification and localization of cellular components , which eAffordability and portability in fluorescence microscopy has been accomplished by retrofitting optical elements  and 3D-printed parts to smartphones cameras ,11\u201313. HS2 Table for a comparison with other different miniature microscopes). After pieces are 3D-printed and a PCB manufactured, the microscope can be assembled in 10 min (see S1 Video) with the capability to acquire images in bright-field and in three fluorescence channels. We present instructions for its assembly, characterization of its illumination homogeneity in each of the fluorescence channels, and demonstrate its operation in a cellular biological assay where it is possible to track single cells captured in microwells.To solve some of these issues, we introduce a portable, low-cost multicolor fluorescence microscope the size of a cube with a side length of 7 cm. Its operation and framework are inspired in the mechanism of a nut and a bolt. The microscope is assembled from only six 3D-printed parts, 4 power light-emitting diodes (LEDs), an eight-megapixel (MP) complementary metal\u2013oxide\u2013semiconductor (CMOS) camera, a metallic rod, and a Printed Circuit Board (PCB) with basic electronics. Aside from the Foldscope , which dS1 Fig. Dimensions of the microscope tube are shown in S2 Fig. The parts are made of black polylactic acid (PLA) and printed with the following settings: speed in X and Y axis of 40 mm/s, temperature of 230\u00b0C, layer thickness of 200 \u03bcm, and infill of 20%. Once fabricated, pieces were manually assembled; see S1 Video for a step-by-step assembly.Microscope parts were designed in Inventor  and fabricated in a 3D printer (Makerbot Replicator 2); see S3 Fig and S1 Table, respectively.The optical system relies on the Raspberry Pi Camera Module V2 that comes with an 8-MegaPixel CMOS image sensor camera and a plastic lens. This lens comes with the camera and no further specifications are provided on it; however, US Patent 7,564,635 B1 provides the description of a lens that has the same focal length (3.04 mm) that is provided in the specification of this camera module. High brightness LEDs  were used to illuminate the sample. Plastic color filters (Roscolux) or single-band pass optical filters (Zeiss) were used as emission filters and placed between the image sensor and the lens. A custom printed circuit board (PCB) containing the electronic components to control the LEDs were placed below the camera. The schematic and the list of components can be found in S4 Fig).A single-board microcomputer (Raspberry Pi 3 Model B) was used to program and control a graphical interface using Python 2.7 and OpenCV. The GUI allowed to acquire images and control the LEDs and different image parameters, such as the exposure time, brightness and contrast  cells were permeabilized with Tween-20  0.05% in Phosphate-Buffered Saline (PBS) 1X for 15 min, centrifuged at 1,200 rpm for 5 min, and washed with PBS 1X. Next, cells were incubated for 20 min either with 4\u2032,6-diamidino-2-phenylindole  or Calcein-AM , or for 10 min with Ethidium Homodimer . The cells were washed again with PBS 1X and centrifuged at 1,200 rpm for 5 min. Then, 20 \u03bcL of the cellular suspensions were spread over three different clean coverslips and allowed to dry. Finally, 10 \u03bcL of glycerol were added and a second coverslip was placed on the top of the samples. For Calcein-AM staining, cells were not permeabilized, and PBS 1X was used instead of glycerol. Finally, coverslips were sealed with enamel.E. coli lipopolysaccharide . Both solutions contained Sytox Orange  at 5 \u03bcM for nucleic acid staining. Devices made of polydimethylsiloxane (PDMS) were fabricated by soft-lithography and consisted of microwells . Cells were placed in three devices, one of them was placed on the stage of the miniature microscope with the stimulus solution and the other two on an inverted fluorescence microscope . 20 \u03bcL of the cell suspension was added to the three devices. After 5 min, 20 \u03bcL of the stimulus were added to two devices, one in our microscope and the other in the Zeiss microscope. 20 \u03bcL of HBSS buffer was added to the other device sitting on the Zeiss microscope, which served as a negative control. Next, images in bright-field and red and blue fluorescence channels were acquired every 10 min in both microscopes. Images were analyzed using an image processing software (Fiji) where fluorescence intensity was measured over time for all individual microwells.Purified neutrophils were donated by Dr. Alejandro S\u00e1nchez Gonz\u00e1lez . Briefly, human peripheral-blood neutrophils were purified from human blood by a non-continuous Percoll density gradient. After purification, neutrophils nuclei were stained with Hoechst 33342  at 16.2 \u03bcM for 5 min. Neutrophils were stimulated with either HBSS buffer 1X (control) or with A single-channel microfluidic device was designed in AutoCAD  and fabricated by soft-lithography . The dev5 cell/mL. Another block of PDMS was used to seal the chamber from the top. The cell chamber was placed on the miniature microscope stage and the whole microscope inside a cell culture incubator at 37\u00b0C with 5% CO2. The bottom of the chamber was manually focused, and the miniature microscope was set in time-lapse mode on the GUI. Next, all cables were disconnected, except for the power cord. During the first ~24 hours, bright-field images were acquired every 15 min. For the last ~2 hours, images acquisition was set to every minute.THP-1 cells (kindly donated by Dr. Vianney Ortiz), were incubated using supplemented RPMI-1640 medium  inside a T25 flask before experimentation. A 1 mL chamber was fabricated using a PDMS slab, plasma bonded to a cover slide and exposed to ultraviolet (UV) light for 3 hours. 900 \u03bcL of fresh media was added to the chamber followed by 100 \u03bcL of THP-1 cell suspension at 100 x 10S2 Table). It also had to offer sufficient accuracy to focus on microscopic objects while being mechanically stable and indifferent to vibrations; but also had to be assembled using 3D printed parts to make it affordable and deployable in low-resources settings. Importantly, our objective was not to develop a fluorescence microscope that offered the same illumination and image quality as commercial microscopes, but rather an easily assembled microscope and affordable for low-budget laboratories. It also had to offer good resolution and image quality for most biological applications, to enable quantitative biological measurements in bright-field and in fluorescence channels.Our goal was to engineer a miniature multicolor fluorescence microscope of simple design that could be quickly assembled with the least possible number of pieces , but also supports the samples mounted on glass slides and serves as a lid for fluorescence observations . The microscope base (bolt) is fixed while the stage rotates to bring the object into focus. It is important to realize that because the microscope is made of PLA, the threading on the nut and bolt can wear out over time (around 4 months) and thus decrease its ability to finely focus on microscopic objects. Nevertheless, the fabrication simplicity of these pieces, enables its easily replacement with new 3D printed parts for less than $1. One disadvantage of this arrangement is that the object under observation is rotated with the stage, yet once focused the object can be manually lifted from the stage and rotated to preserve the orientation.The microscope is inspired in the simple mechanism of a nut and bolt, S2 Table), making them unsuitable for low-cost applications. Because LEDs have a very narrow band spectrum (20\u201340 nm), it is possible to omit the detection cube and just keep the emission filters. With these considerations, bright-field mode is enabled in our microscope using a white LED placed on the center top of the lid , while fluorescence observations are achieved using 3 high-power color LEDs  that illuminate the sample from the sides . Three plastic emission filters are placed on a hollowed-out tray that slides on the shank of the microscope, keeping one of the hollows empty for bright-field observations. However, it is possible to use regular microscope emission filters by modifying the dimensions of the tray and the shank. In summary, the simplicity of our configuration contrasts with other microscopes that require several 3D-printed parts [Most fluorescence microscope configurations use fluorescence detection cubes  incorporated into a filter wheel. These detection cubes have excellent optical qualities, but the cost for single-fluorescence channel filter cube can exceed a few hundred US dollars , in a similar configuration to what others have reported [The microscope objective consists of a plastic lens detached from a commercial camera module and placed upside-down 25.4 mm above an 8 MP CMOS sensor , a metallic rod, 4 high-power LEDs, a CMOS camera, a lens, and an electronic control unit that powers the LEDs from a single 9V power supply, see  ~9.5 kg , togetheS4 Fig.To operate the microscope, we created a graphical user interface (GUI) in Python 2.7 that allowed us to: (i) turn on and off the different color LEDs on demand, (ii) set the exposure time, (iii) capture still images, (iv) take videos or time-lapse images, and (v) save images in different formats, among other functions, Fig 2 shows the step-by-step operation of the microscope. It involves placing the sample on the stage by opening the lid. Next, the lid is closed to isolate the sample from external light sources. For bright-field observations, the white LED is turned on from the GUI and the tray is shifted to position the empty hole on the optical path. The stage is manually rotated clockwise or counterclockwise to focus the sample. For fluorescence observations, one of the color LEDs is turned on while the filter tray is manually slid to match the corresponding emission filter. S2 Video shows how easily the microscope is operated by a user.The theoretical magnification of our microscope is obtained using the magnification formula , conside2 (525 \u03bcm x 394 \u03bcm). According to the Rayleigh criteria, the theoretical optical resolution calculated at a wavelength of 550 nm is 0.448 \u03bcm. The focal length and working distance of the lens are ~3.05mm and ~0.1 mm, respectively. The lens has a numerical aperture of 0.45 and a depth of field of 1.46 \u03bcm. To determine the optical resolution of our system, we measured the full width at half maximum (FWHM) of the point spread function (PSF) for forty 1-\u03bcm fluorescent beads (Firefli Fluorescent Blue) spread over the whole field of view. The estimated FWHM of the 40 microbeads with our microscope is 1.187 \u03bcm, identical to the one obtained with a 20X objective using a Zeiss microscope, Fig 3A and 3B. We also imaged a 1951-USAF resolution target . As seen in  our microscope can clearly solve two lines separated by 2.2 \u03bcm. This is very similar to the calculated smallest resolvable line pair spacing of 2.05 \u03bcm, obtained by dividing the total display magnification (257.3x) by two times the monitor pixel size (0.264 mm) [The field of view is ~0.2 mmM, a measurement of contrast on gratings [M \u2265 0.1 [Fig 3D, the center of the image produces sharper contrasts for all the line sets, although there is a pronounced decreased of CM values on the left side of the image, possibly due to the inclination of the sample stage of the microscope. Nevertheless, all the line sets of the USAF target produce a CM > 0.3, even for patterns as small as 2.2 \u03bcm, a sufficient resolution for most cell-based measurements [A common issue with custom-made microscopes is the radial loss of resolution farther from the center . This isgratings ), along [M \u2265 0.1 . As showurements ,13,16 anTo correct for uneven illumination intensity from light sources, fluorescence microscopes employ the Koehler illumination, a set of lenses positioned between the light source and the sample. The simThus, we wondered whether direct illumination, where the light source  is placed near the sample, could give us a uniform illumination. This is a simpler and inefficient configuration as only a small percentage of the light produced by the LEDs reaches the sample , but becFig 4 shows the heatmaps of the images captured by the CMOS color sensor at these different angles and considering only the digital channel closest to the emitted light. Data from the contribution of the rest of the RGB components can be found in . As it can be observed, vignetting is appreciable in most cases, but it is more manifested at a 0\u00b0 angle. However, as the angle increases to 22\u00b0, this vignetting is reduced, and almost eliminated for the red (R) channel and to a lesser extent on the green (G) channel, but is still significant for the blue (B) channel, noticing a reduced illumination on the left side of the heatmap, opposite to where the UV LED was placed. Increasing the angle to 45\u00b0 reduces this vignetting for the B channel compared to the other angles; however, for the R channel the left side is less illuminated than the right side, while the G channel shows a higher radial darkening than at 22\u00b0. Emission detection on the other channels is highly attenuated but not completely blocked; this can be attributed to the poor optical quality of the plastic filters. Others [Because the color LEDs could not be placed on top of the sample, as it was already occupied by the white LED, the only positions left where the LEDs could be positioned were in the corners of the lid or at the cardinal points; we decided on the latter as it facilitated the mechanical design. We investigated how uniform was the illumination by illuminating fluorescent solutions  at angles of 0\u00b0, 22\u00b0 and 45\u00b0 with a 1 s exposure times and employing the plastic filters (we fabricated three different lids to perform this experiment). . Others  have rep. Others .Fig 5A shows micrographs of THP-1 cells stained with EthD-1 (ex/em 528/617 nm), Calcein-AM (ex/em 496/516 nm) and DAPI (ex/em 360/460 nm) using plastic filters. The intensity profile across the diameter of two cells for the three-fluorescence channels after subtracting background intensity is plotted in Fig 5B. As can be observed, plastic filters block fluorescence bleed-through from neighboring channels. Also, there is no significant difference with commercial glass filters, S8 Fig. This experiment demonstrated that our fluorescence microscope produces sufficient image quality to detect and analyze single cells.To demonstrate the utility of our microscope to image single mammalian cells, we acquired bright-field and fluorescence images using plastic filters (<$1) and optical glass filters (>$100). To demonstrate the utility of our microscope in quantitative biological experiments, we performed a fluorescence time-lapse experiment to track the production of neutrophil extracellular traps (NETs) from single cells . The nucFig 6A), and with a high-end inverted fluorescence microscope for comparison (not shown). As can be appreciated, it is possible to distinguish single cells trapped in each well. Analysis of individual wells captured with our microscope is shown in Fig 6B, where each gray trace corresponds to the fluorescence intensity of one individual well and the thick colored curves indicates the average of ~170 wells. The results for Sytox Orange with both microscopes have similar trends: in both cases, most of the wells showed a gradual increase in fluorescence intensity after ~15 min, reaching a peak at ~50 min (an indication of loss of plasma membrane integrity and thus yielding an indirect measurement of NETs formation), subsequently slowly decreasing as the DNA diffused out of the wells. In the case of Hoechst channel, the results of our microscope compare favorably to the Zeiss microscope, with both data showing a slight fluorescence intensity from the beginning of the assay\u2014as all the cells nuclei were stained\u2014increasing gradually to a maximum intensity at 50 min, to eventually decrease afterwards. A negative control experiment, where cells were incubated with Hank\u2019s solution, showed no change in fluorescence intensity over time, S9 Fig. The slight difference in the data obtained with the Zeiss microscope\u2014higher peaks and smother curves\u2014was expected given the high quality of its optical system, while ours had a higher background noise. Overall, it is possible to perform quantitative biological experiments in our microscope, enabling fluorescence time-lapse microscopy.Bright-field and fluorescence images (UV and Red) were acquired every 10 min for 2 h with our microscope . Fig 7C shows a bright-field micrograph of a single cell flowing in the microfluidic channel, and Fig 7D shows fluorescence micrographs of two cells acquired with a 100 ms exposure time. S3 Video shows the facility to focus the optics onto the microfluidic channel and to start recording the cells flowing through the channel, demonstrating the capabilities of our fluorescence microscopy system to not only capture still images of objects sitting on a microscope slide.Another feature of our miniature microscope is the capability to record video in bright-field and fluorescence mode. Using a single-channel microfluidic device with a width of 40 \u03bcm and a height of 20 \u03bcm, we injected THP-1 cells stained with Calcein-AM at a speed of ~150 \u03bcm/s, and recorded a video while cells flowed through the channel  the lamellipodia of cells extending and retracting, (ii) the migration of cells over time, and (iii) cells undergoing division.2 incubator), but we also foresee applications in diagnostics or telemedicine. Because of its low-cost and size, several microscopes could be assembled to monitor several assays at once.Fluorescence microscopy is an important instrument in biomedical research. Here, we have demonstrated that using 3D printed parts and basic electronic components it is possible to build a miniature 3-channel fluorescence microscope for under $100. In our design, we favored simplicity over other metrics so that it could be assembled rapidly (10 min), although still able to produce sufficient image quality to analyze single cells. We demonstrated that placing color LEDs at different angles from the sample produces a homogenous illumination. We also showed that plastic filters minimized fluorescence bleed-through to the same level of optical glass filters. To demonstrate its application in single cell assays, we monitored the production of NETs from single neutrophils, yielding similar data to a commercial microscope. Our microscope is ideal for downstream applications using microfluidic devices, as demonstrated here, or in situations where space is a premium , our microscope is suitable for long-term experimentation which could enable acquisition of large amounts of data and translate into a better characterization of biological systems. One of the limitations of our microscope is its narrow field of view and its fixed magnification. Thus, an area of opportunity is to develop a motorized stage or use larger CMOS image sensors. Another limitation is the possible wear-out of 3D printed parts; however, all the microscope parts could be fabricated using sturdier materials, such as aluminum or other thermoplastics, using a variety of fabrication techniques. Albeit these limitations, our microscope performed a fluorescence time-lapse experiment of single cells yielding similar data to a conventional microscope.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 Videohttps://github.com/BioARTS-Lab/Miniature_Microscope/blob/master/Supporting%20Information/S1_Video.mpg.(MPG)Click here for additional data file.S2 Videohttps://github.com/BioARTS-Lab/Miniature_Microscope/blob/master/Supporting%20Information/S2_Video.mpg.(MPG)Click here for additional data file.S3 Videohttps://github.com/BioARTS-Lab/Miniature_Microscope/blob/master/Supporting%20Information/S3_Video.mpg.(MPG)Click here for additional data file.S4 Videohttps://github.com/BioARTS-Lab/Miniature_Microscope/blob/master/Supporting%20Information/S4_Video.mpg.(MPG)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 Fig(Left) Front view of the mechanical tube of the microscope. Thread pitch is 1 mm. (Right) Cross-sectional view of the mechanical tube, showing the lens attached to the top of the tube. All dimensions are in mm.(TIF)Click here for additional data file.S3 Fig(a) Circuit design. (b) Printed circuit board layout.(TIF)Click here for additional data file.S4 FigThe GUI was coded in Python by combining Tkinter, picamera and OpenCV libraries.(TIF)Click here for additional data file.S5 FigEach row corresponds to a different LED; the angle and the digital color channel analyzed are indicated on the top right corner. R: Red, G: Green, B: Blue. Fluorescence intensity has been normalized to a maximum value of 1.(TIF)Click here for additional data file.S6 FigEach row corresponds to a different LED; the angle and the digital color channel analyzed are indicated on the top right corner. R: Red, G: Green, B: Blue. Fluorescence intensity has been normalized to a maximum value of 1(TIF)Click here for additional data file.S7 FigEach row corresponds to a different LED; the angle and the digital color channel analyzed are indicated on the top right corner. R: Red, G: Green, B: Blue. Fluorescence intensity has been normalized to a maximum value of 1(TIF)Click here for additional data file.S8 FigBright-field and fluorescence micrographs captured with our microscope using plastic filters (left) or Zeiss filters (right). THP-1 cells stained with different fluorochromes: EthD-1 (red), Calcein-AM (green) and DAPI (blue). Images using all filters were acquired for each fluorochrome. Graphs show intensity profile of two cells across all channels, showing that there is no fluorescence bleed-through between channels.(TIF)Click here for additional data file.S9 FigTraces of fluorescence intensities from single wells for the negative control experiment shown in (TIF)Click here for additional data file.S10 Fig(a) Photograph of the inside of a cell culture incubator showing the microscope and the microcomputer Raspberry. (b) Photograph of the cell culture chamber made of PDMS.(TIF)Click here for additional data file."}
+{"text": "Data generated from a system of interest typically consists of measurements onmany covariate features and possibly multiple response features across allsubjects in a designated ensemble. Such data is naturally represented by oneresponse-matrix against one covariate-matrix. A matrix lattice is anadvantageous platform for simultaneously accommodating heterogeneous data types:continuous, discrete and categorical, and exploring hidden dependencyamong/between features and subjects. After each feature being individuallyrenormalized with respect to its own histogram, the categorical version ofmutual conditional entropy is evaluated for all pairs of response and covariatefeatures according to the combinatorial information theory. Then, by applyingData Could Geometry (DCG) algorithmic computations on such a mutual conditionalentropy matrix, multiple synergistic feature-groups are partitioned. Distinctsynergistic feature-groups embrace distinct structures of dependency. Theexplicit details of dependency among members of synergistic features are seenthrough mutliscale compositions of blocks computed by a computing paradigmcalled Data Mechanics. We then propose a categorical pattern matching approachto establish a directed associative linkage: from the patterned responsedependency to serial structured covariate dependency. The graphic display ofsuch a directed associative linkage is termed an information flow and thedegrees of association are evaluated via tree-to-tree mutual conditionalentropy. This new universal way of discovering system knowledge is illustratedthrough five data sets. In each case, the emergent visible heterogeneity is anorganization of discovered knowledge. Nearly all scientific researches are geared to acquire knowledge and understanding onsystems of interest. So data generated from a target system typically consists ofmeasurements on many covariate features and possibly multiple response featuresbelonging to subjects, who constitute a representative ensemble of the system. Assuch a system data set typically consists of one response data matrix against acovariate data matrix. These two matrices share the common ensemble of subjects,which are arranged along its row-axis, while their own features are arranged alongtheir own column-axis, respectively. The matrix lattice is indeed an advantageousplatform for revealing patterned structures, particularly for dependency withinresponse or covariate sides. Moreover these two platforms become the jointfoundation for all the directed associative linkages going from response side tocovariate side.It is known that, among these subjects, whether they are human, animal or plants oreven cells, are likely interconnected, and among these relevant features, no matterthey are on either response or covariate sides, are interrelated. Suchinterconnections and interrelatedness also weave interacting relations betweensubjects and features. Thus, as a rule, each system data set is expected to containthese three fronts of dependency, which have unknown detailed structures, and waitto be explored and discovered. Further we conceive the system understanding andknowledge as the linkages going from response\u2019s dependency structures to covariate\u2019sdependency constructs.A guideline for successfully extracting system understanding and knowledge was indeedlaid more than four decades ago in physics. The Physics Nobel laureate P. W.Anderson , in his In this paper we propose a protocol for analyzing any system data set. A quickoverview of this protocol is given as follows. First, we adopt unsuperviseddata-driven computing, which is free from any unrealistic structural ordistributional assumptions, in order to effectively capture authentic information ofdependency structures and constructs. Secondly, we employ a graphic display platformto arrange and represent extracted information in order to stimulate understandingand explore knowledge regarding the system under study. The guiding principleunderlying such a graphic display platform is appealing to the formidable capabilityof visual processing in man .Our protocol embraces a simple, but distinctive concept: a system likely containsmultiple system mechanisms in both response and covariate sides. To embrace thisconcept in a natural fashion, a nonlinear association measure is evaluated upon eachpair of features on the response and covariate sides. We then apply an Ultrametricclustering algorithm to partition the collection of covariate features into acomposition of synergistic feature clusters (or groups). Likewise the collection ofresponse features are partitioned. Each synergistic feature group functionallyreveals a distinctive pattern of dependency, so indeed represents a distinctmechanism of the target system. Therefore, we need to seek for interpretationpertaining to a single response mechanism through its linkages to a series ofcovariate mechanisms. A computational algorithm coupled with a graphic displayplatform is developed to make such linkages explicitly interpretable and pictoriallyvisible. It is worth emphasizing that system understanding and knowledge involvedwith multiple mechanisms in multiple different ways. Also it is evident to note thatthis composite-level concept fundamentally and contrastingly distinguishes this dataanalysis protocol from statistical modeling and supervised learning.In contrast, traditional statistical modeling and popular supervised learning sharethree common key characteristics: 1) primarily accommodate only one single responsefeature at a time, which destroys the entire response dependency; 2) utilizesconditioning argument on covariate features, which ignores covariate dependency andpotential involvements of multiple mechanisms; 3) and imposes independence amongsubjects, which imposes unrealistic homogeneity. At the end, data-analysis resultsare pushed through the likelihood principle or an optimizing process with respect toa man-made criterion.Further model-based techniques only accommodate selective data types, in otherswords, a data type often becomes the decisive factor in choosing models. For abinary response feature, the logistic regression model is the definite choice. For acontinuous response feature, the linear regression is the definite choice. Thelogistic and linear modeling frameworks break down when the response feature hasmore two categories. When two or more response features are of interest, statisticalmodeling break down as well. The latter, so-called multiple response issue, wasraised more than half century by John Tukey . Up to tbasically re-normalizing all features into categorical ones sharinga common digital-coding range. A real-valued feature is re-normalizedinto a digital-categorical via its own possibly-gapped histogram ..6]. DataWhen these two marginal clustering trees resulted from the DM computations aresuperimposed respectively on the two axes of the matrix, visible multiscalepatterned-blocks emerge on the permuted matrix lattice, which is consequently termedheatmap. This heatmap collectively reveals a detailed version of mechanism-specificstructural dependency by showing the three fronts of information contents containedin the data matrix: 1) how and why some subjects group closely together, and how andwhy they are far away from other clusters of subjects; 2) how and why theUltrametric tree on features represents and constitute a map of key factors of thesystem; 3) how and why the multiscale block-patterns bring out the heterogeneity ina collective fashion through the interacting relational characteristics betweensubject and features.We further construct a directed associative linkage from one heatmap pertaining to aresponse mechanism to another heatmap pertaining to a covariate mechanism. Theconstruction via a graphic display exhibits that such a linkage maps the membershipsof a clustering composition taken from the response\u2019s clustering tree on subjects(row-axis) onto a clustering composition taken from the covariate\u2019s clustering treeon subjects. That is, based on the two trees, each subject is encoded with a pair ofcategorical code: one from response side and the other from the covariate side.Therefore, the strength of such a linkage is again evaluated by the directedconditional-entropy based on the combinatorial information theory. This graphicdisplay in fact facilitates the interpretation of the linkage by matching multiscaleblock-patterns of the response mechanism to the multiscale block-patterns of thecovariate mechanism. So the interpretation is explicit, visible and readable. Thisgraphic linkage is called categorical-pattern matching. We then extend this platformof graphic display to accommodate a series of a covariate mechanisms, and term it anInformation flow.X with regardto Y.Evaluation of amount of information conveyed by  In his 1965 paper [\u201c\u2025 It is only important for me to show that the mathematical problems associated witha purely combinatorial approach to the measure of information are not limited totrivialities.\u201dIndeed the combinatorial approach has been well known in Information Theory since C.E. Shannon\u2019s pioneer works  11].Ho.Ho11].In this paper we discuss that the combinatorial approach of information in fact givesus an universal measure of associative relation between two variables. Based onconditional entropy, this relational association concept will be seen as especiallysuitable for unsupervised machine learning and its inferences, in which the\u201csample-to-population\u201d sense is not involved. Along the developing process, we alsoreflect why the linearity backbone of correlation can cause invalid and even oftenmisleading interpretations on real data. Before introducing such a measure ofentropy based associative relation, it is beneficial to review this combinatorialapproach of Information.N(= m \u00d7 n)subjects contained within an ensemble. If these N subjects aredivided in m sub-ensembles of size n, then theequal-potential sampling scheme on N subjects is equivalent tofirst sampling with equal potentials from the collection of msub-ensembles, and secondly sampling one subject with equal potentials from thechosen sub-ensemble of n subjects. This so called composition rule, is exactly equal to H(X) andE[X \u2192 Y] of two featuresdenoted by X and Y, that is, if Xis capable of conveying a non-negligible amount of information in relation toY, or vice versa, then X andY are dependent. After building a mutual conditional-entropymatrix, subsequently, as will be demonstrated in sections blow, an unsupervisedlearning algorithm is applied to perform the task of feature regrouping. That is, asynergistic group of features is seen as constituting a mechanism, while twosynergistic groups being antagonistic would be seen as two separate mechanisms.Unlike the logistic and linear regression models in statistics, our proposedcomputational protocol will link one synergistic response-feature group to one orseveral synergistic covariate-feature groups. This proposal interestingly fulfillsTukey\u2019s postulation of appealing to Taxonomy and classification methodologies on themultiple response issue ) of being nearzero. Via this heterogeneity, scientists specifically figure out how themeasurements of a synergistic response-feature group upon a subject-cluster can beexplained collectively by multiple distinct series of block-patterns manifested byserial synergistic covariate-feature groups upon a partition of the originalsubject-cluster. From this perspective, our data analysis is clearly fundamentallydistinct from convention statistical modeling and supervised learning methods, whichheavily rely on the hypothesized sample-to-population homogeneity.Here a block-pattern is taken as a knowledge locus, and a linkage via theexclusiveness is meant to be equipped with an extreme conditional entropy as follow:1) primarily dominant by Redcolor-coded subjects (Y = 0); 2) purely Blue color-coded subjects(Y = 1); 3) primarily dominant by Blue color-coded subjects(Y = 1); 4) a mixture of Red and Blue color-coded subjects. Itis surprising that by allowing heterogeneity, the misclassification result of aLogistic regression with a given threshold can be very much improved and moreprecisely understood. This hierarchical clustering tree provides an extra advantagethat there is no need to choose an ad hoc threshold to count for the false-positive(FP) and false-negative(FN).Apart from being not able to accommodate a response variable beyond binary responsevariable, a Logistic regression also critically suffers from its linearity imposedconstraint of homogeneity. It can\u2019t accommodate heterogeneity such as shown in theHowever, the heterogeneity through the hierarchical clustering tree ofPossibly-gapped histogram based re-normalization Let n \u00d7m data matrix with n subjects being arrangedalong the row-axis and m features along the column-axis. Eachfeature specific column has to undergo a digital re-normalization procedure based ona data-driven possibly-gapped histogram as illustrated in m features are mixed in data-types: continuous,discrete and categorical, as would be seen in the Heart data below, there-normalization becomes a rather tricky issue to be resolved in order to achieve alarge degree of uniformity. Here we suggest a guideline: two features withrelatively low mutual conditional-entropy should be similarly digitallycoded. Denote the n \u00d7 m re-normalizeddata matrix be However, when the Synergistic-vs-antagonistic feature grouping via Data Cloud Geometry (DCG)algorithm. Upon this n \u00d7 mre-normalized data matrix m \u00d7m mutual conditional-entropy matrix, say \u039e, for allfeature-pairs , which is generic bivariatedigital coding, i.e. two separate columns of x \u2208 {a,b, c, \u2025} and y \u2208{A, B, C, \u2026} according tonotations in H(Y|X), is evaluated withrespect to the discrete distribution of X, while the entropyE(0)(Y), also conventionallydenoted by H(Y), is calculated with respect to thediscrete distribution of Y.m \u00d7 m mutual conditional-entropy matrix \u039ecan be taken as a distance matrix for feature-grouping computations. TheData-Cloud-Geometry (DCG) computing algorithm employed here aims at building anUltrametric clustering tree, say Then the uting in  8]..m \u00d7 m muDCG algorithm: We begin with taking mutual the conditional entropymatrix \u039e = [dij] as a distance matrix.In general a distance matrix is derived through a distance measure, which istypically an empirical choice of system scientists. T, whichis typically chosen with respect to the histogram of all entries of \u039e =[dij], a similaritymatrix is generated as Step-1 With respect to a temperature  ST(\u039e) is normalized by itsrow sum. So ST(\u039e) becomes atransition probability matrixPT(\u039e).Step-2 Then each row ofPT(\u039e) gives rise to aregulated Markov random walk, which starts randomly from a leaf-node,and then removes a leaf-node, when its number of visits by this Markovrandom walk has gone beyond a threshold. When a leaf-node is removed,the transition matrix PT(\u039e)is regenerated by deleting the corresponding row and column. This stepis designed to keep the Markov random walk from being trapped in a localregion of the data cloud.Step-3 T.Step-4 A trajectory of a regulated random walk will give rise to aleaf-node-removal recurrence time series, which is equipped with severalspikes, which indicating the random walk has just enters a new,unexplored local region. Therefore, all leaf-nodes removed between twosuccessive spikes are taken as being in the same cluster with respect tothe temperature i, j) entry is coded 1 if thei\u2013th and j-th leaf-nodes are inthe same cluster. An ensemble of such trajectories will give rise to anensemble of such cluster-sharing matrices, which is then summarized intoa matrix of cluster sharing probability, denoted asEn[PT(\u039e)]. \u2264 maxd,d.)Step-5 So a trajectory will give rise to a binary matrix: its(En[PT(\u039e)]is taken as the number of clusters, sayN(T), being present in thetemperature scale T, while the explicit clusteringcomposition can be extracted by applying HC algorithm, or otherclustering algorithm based on N(T) clusters.Step-6 The number of significantly non-zero eigenvalues ofN(T) againstT . We choose a temperatureTi from eachleveling-off or constant segment of this plot.  Denote this set of selected temperatures as{T1, \u2026,TK} and theircorresponding clustering compositions Step-7 Plot By superimposing this Ultrametric tree on row and column axes of \u039e, its framed matrixlattice will naturally show multiscale block-patterns, as illustrated in panel(A) ofHere it is worthy clarifying and reiterating the conceptual nature of two features orfeature-groups being \u201csynergistic vs antagonistic\u201d. These two contrasting conceptssimply refers to increasing or decreasing \u201cpotentials\u201d of pattern formation ofdependency between the two features or feature-groups. As \u201cdependency\u201d is naturallyrevealed through \u201ccategorical\u201d correspondence, which is typically visible when twofeatures or feature-groups are put together side-by-side under an unsupervisedlearning setting. The strong categorical correspondence is exactly the phenomenonconveyed by being synergistic, and is precisely captured and measured by having lowmutual conditional entropy. Specifically they are \u201ccategorically\u201d corresponding toeach other in a way that, by knowing a category of one feature upon a subject, wecan predict very well about which category of the other feature upon the samesubject will belong to.In contrast, this good predictability disappears when two features are indeedantagonistic. That is, two antagonistic features are lacking \u201ccategorical\u201dcorrespondence, so not only patterns of dependency can hardly emerge when they areput together side-by-side, but also they in fact tend to destroyed patterns ofindividual feature or feature-groups. This potential of destroying patterns is what\u201cantagonistic\u201d is referring to.Furthermore we remark that the synergistic and antagonistic correspondences can behighly non-linear. However, if such correspondences are in fact linear, then thesynergistic correspondence is equivalent to correlations going either highlypositive or highly negative, while the antagonistic correspondence is equivalent tonearly zero correlation.Data mechanics on matrix data Features sharing a synergisticfeature-group are highly dependent because they potentially share the same mechanismwithin the study system. Such dependency will allow unsupervised learning algorithmsto more effectively reveal fine scale interacting relational patterns betweensubject-clusters and feature-clusters. That is, the row-axis of distance metrics are updatedwith respect to the tree structure obtained from the previous step on the otheraxis. Denote the final two Ultrametric trees The unsupervised learning algorithm employed here is called Data Mechanics (DM), seeFushing and Chen (2014) and Fushing et al. 2015). Data Mechanics is an iterativealgorithm that build one DCG-based Ultrametric tree on row-axis and one oncolumn-axis alternatingly. In each iteration, . Data Meuting in .Data Mechanics:mrows of Step-1 We adopt the Euclidean distance measure on all N* clusters.Consider each column being extended with N* extradimensions of average among member-rows of clusters of n columnwith m + N* dimensional Euclideandistance.Step-2 Select a tree level on Step-3 Based on distance matrix Step-4 Based on one selected level of the tree Step-5 Repeat the Step-2 to Step-4 for two or three times, or until bothtrees Step-6 Let the final two marginal trees are denoted as The uniformity within each of the finest scale block collectively forms thestochastic structures contained within Organization of knowledge via information flows Now consider an \u00d7 mRe responsedata matrix, n \u00d7mCo covariate data matrix[Algorithmic steps for discovering and confirming information flows:][Re-normalizing all features]: Matrices [Re-grouping synergistic features]: Upon mRe \u00d7mRe mutualconditional-entropy matrix \u039eRe is computed,so is mCo \u00d7mCo mutualconditional-entropy matrix \u039eCo based on[Discovering block-patterns via Data Mechanics]: Each datasubmatrix of [Exploring information flows via Categorical-patternmatching]: Each row-marginal tree E[Y \u2192 X]. A lowvalue of such directed conditional entropy implies that there existstrong associative patterns. Specifically a strong associative patternis identified as a cluster or branch of [Information flows]: Organize all associative patterns withrespect to a series of coupling geometries via a series of heatmaps.This graphic display is called Information flow, which is taken as arepresentation of knowledge from one response mechanism to a series ofcovariate mechanisms.Confirming an information flow and calculating its error rate. Due tothe exploratory nature of an organization of directed associative patterns, a resultof categorical-pattern matching, needs a formal confirmation, and then its errorrate has to be evaluated. All these computations are scale-dependent, but rathersimple and elementary. Here the scale-dependence is referring to a clusteringcomposition of subjects pertaining to a chosen tree level of the row-marginal treeGiven a pair of clustering compositions respectively coming from response andcovariate sides, observed directed conditional entropies are evaluated on eachindividual cluster of the clustering composition on the covariate side, asillustrated in For an error rate of an information flow with only one covariate synergisticfeature-group, either majority rule or randomized rule can be applied within eachcluster on the covariate side to calculate individual cluster\u2019s error rates, andthen a weighted overall version is calculated with respect to sizes proportions ofthe covariate clustering composition. The reason underlying this simplicity is givenas follows. Randomly select one subject and remove its cluster membership on theresponse side. The key is that based on the semi-unsupervised learning paradigm,this subject\u2019s row covariate vector needs to participate in the construction ofrow-marginal tree For an error rate of an information flow on serial covariate synergisticfeature-groups, the overall error rate is calculated in a weighted fashion. Theweighting should be inversely proportional to their individualconditional-entropies. Such simplicity is one significant advantage of adopting anunsupervised learning paradigm. That is, here there is no need to perform thecross-validation as needed in supervised learning paradigms.In this section we analyze five simple system data sets from UCI Machine LearningRepositoryhttps://archive.ics.uci.edu/ml/datasets.html). Each data set ischosen for idiosyncratic reasons and characters: 1) the 1st data set with 1D binaryresponse feature is to show why an information flow is more advantageous overLogistic regression model; 2) the 2nd data set with 1D continuous response featureis to recognize the fact that a data set can only sustain limited, not fullspectrum, of resolutions of information content as implied by a linear regressionmodel; 3) the 3rd and 4th data sets deal with multiple response features withdistinct data types; 4) the 5th data set consists of covariate features of alltypes: from continuous, discrete to categorical ones, in which all features need tobe properly digitally coded.was to obtain a series of reconstruction formulas to predict brain weight givenmeasurement of head size. It is clear that, based on associative patterns of thethree features via in the information flow shown in As another demonstration of how dependency structures work, we classify theassociation between gender and head size into four groups :1) G4;2) G8; 3) G5-G7; and 4) G1-G3, as shown in panel (F) of Electricity data The data from  The mutual conditional-entropy matrix shows two synergistic feature groups in Further, via simple random sampling without replacement scheme, the classificationperformance pertaining to two scales of clustering compositions: 3 clusters (Yellowcolor-coded boxes) and 9 clusters (Black color-coded bars), in the information floware evaluated through 1000 simulations and presented in box-plots of 95%, as shownin In this paper we develop one universal platform: algorithmic computing protocol plusgraphic display techniques for system data analysis. Our computing protocol isdeveloped under the guiding principle of having multiple synergistic mechanismscontained in a system. Our goal is geared to first extract authentic informationcontents contained in a system data set. And secondly ourcategorical-pattern-matching via graphic display is to stimulate properunderstanding of computed information, and thirdly to discover pertinent knowledgeabout the system under study. The resultant system knowledge on one single responsemechanism is visible and explainable through a series of covariate mechanisms. Suchknowledge is organized and represented through one single information flow. And asystem is likely better understood by multiple information flows.An information flow functionally maps the response\u2019s structural dependency intocovariate\u2019s patterned dependency. These dependency patterns and structures arecomputed through Data Mechanics on data matrix, and are collectively revealedthrough multiscale blocks framed by a clustering tree superimposed on a group ofsynergistic features and another clustering tree on the ensemble of subjects. Thatis, the dependency patterns and structures summarize essential information contentson response and covariate data matrices, respectively, without involving potentialdistortions possibly caused by unrealistic modeling or distribution assumptions. Inthis era of big data, we are confident that our universal platform of system dataanalysis has high potential merits in sciences.In contrast to our universal platform, it is worth mentioning and discussing thenarrow perspective tied to model selection techniques in statistics. On top ofemploying ad hoc and narrowly focused criterions, like sum of squared error (SSE),all model-selection techniques choose only one set of covariate features from afixed ensemble of potential models, and ignore all potential groups of features,which might result in a just slightly larger SSE than the minimum one. It seems likethat no extra information can be offered from the second best sets of covariatefeatures. This is likely totally not true. Since there might exist several distinctand meaningful mechanisms simultaneously associating with one single dimensionalresponse feature. For instance, consider a study on causes of a behavioral disorder,such as autism or obesity. Wouldn\u2019t the potential causes of a disorder become moreand more complex when more than more subjects are included into the study? Moresubjects certainly will bring in more diverse and different psychological factors,environmental conditions, cultures and genetic makeups and many others. These causalfactors surely tightly tangle together as multi-faces of the disorder.Further all model selection techniques assume an implicit fundamental assumption thatthe ensemble of potential models is invariant with respect to the number ofinvolving subjects. Like the conditioning argument in all regression analysis, thisinvariance assumption is another strong evidence of ignorance of structuraldependency on the covariate side. To be more specific, as the ensemble of observedsystem subjects becoming larger, the subject\u2019s \u201ccommunity structure\u201d would be morefully exposed. That is, distinctions and gaps among these communities are to be moreevidently expressed through block patterns due to large and fine scales dependencyamong covariate features. The presence of finer and finer scales dependencystructures is exactly the reason underlying the fact that no models are correct whenthe sample size is really big. But this invariance assumption imposed by all modelselection techniques strictly require practitioners to blindly give up the truththat there are potential multiple mechanisms behind trends of one single responsefeature. On the other hand, this critically unreasonable assumption of fixedensemble of potential models disregarding sample sizes also reflects theimpossibility of the issue of how to properly grow the ensemble as sample sizeincrease.At the end, we remark that our directed associative linkage expressed through graphicdisplay can fundamentally resolve the recent issue of reproducibility of researchresults for publications in major scientific journals. The reason is that, eventhough this reproducibility concern has pressured scientists to be more vigilant andrigorous when they conduct and report their data analysis, unintentional or carelessmistakes or human fallacies can still creep into the modeling and affect summarizingparameter values. Requirement of submitting the original data in the submissionprocess for journal publication would only prevent potential human errors to somelimited extents. Since effects of man-made assumptions, particularly involved incomplicate modeling, are still hard to be filtered out and prevented fromcontributing to implications made from reported statistical results.S1 Box(PDF)Click here for additional data file.S2 Box(PDF)Click here for additional data file.S3 Box(PDF)Click here for additional data file."}
+{"text": "The topographic location of acute pontine infarction is associated with clinical syndromes and prognosis. Previous studies focused on isolated pontine infarction, but the topographic location of unisolated pontine infarction has remained unclear.This was a prospective, multicenter, longitudinal registry study. Patients with acute pontine infarction confirmed by magnetic resonance imaging (MRI) were enrolled. Based on the territory of the pontine artery, the topographic location was divided into anteromedial, anterolateral, tegmental, bilateral and unilateral multiple infarctions.From May 1, 2003, to Oct 31, 2017, 1003 patients were enrolled, and 330 had unisolated pontine infarction. For isolated pontine infarction, 44.9, 19.8, 16.0, 13.1 and 6.2% of patients had anteromedial, anterolateral, tegmental, bilateral and unilateral multiple pontine infarctions, respectively. For unisolated pontine infarction, 30.3, 19.7, 24.5, 15.2 and 10.3% of patients had anteromedial, anterolateral, tegmental, bilateral and unilateral multiple pontine infarctions, respectively.In this large series study, our data revealed fewer anteromedial infarctions and more tegmental and unilateral multiple infarctions in patients with unisolated pontine infarction than in patients with isolated pontine infarction. Pontine infarction is the most common type of stroke in the posterior circulation territory and accounts for 7% of all ischemic strokes , 2. TherIn brief, previous studies usually recruited patients with isolated pontine infarction , 3\u20136. InAlthough unisolated pontine infarction is often identified in clinical practice , there iThe research protocol  was reviData were prospectively collected from patients with acute pontine infarction. The patients were enrolled if they met the inclusion criteria as follows: (1) older than 18\u2009years; (2) admitted to the hospital within 7\u2009days of stroke onset; (3) MRI  and magnetic resonance angiogram (MRA); and (4) intracranial and extracranial cerebral arteries visualized by ultrasound, MRA, computed tomography angiography (CTA) or digital subtraction angiography (DSA). The patients were excluded if they met any of the following criteria: (1) missing clinical or imaging information, (2) brain tumor, (3) brain parasites, or (4) no lesion in DWI images.On admission, demographic information (including age and gender), stroke risks  history) and the National Institutes of Health Stroke Scale (NIHSS) on admission were collected. Current drinkers were categorized by heavy intake (more than 14 drinks per week in women or more than 21 drinks per week in men) or episodic heavy intake (more than 5 drinks in 1 episode at least once per month) . Blood tAll patients underwent MRI on admission using either a 1.5\u2009T or a 3.0\u2009T MRI unit. Scanning sequences included T1-weighted imaging, T2-weighted imaging, fluid-attenuated inversion recovery sequence, apparent diffusion coefficient maps, DWI and time-of-flight MRA covering the circle of Willis. The stroke subtype was determined based on the DWI images with reference to other sequences.Based on the territory of the pontine artery, pontine infarction has 5 subtypes Fig.\u00a0: anteromt-test or analysis of variance (ANOVA) was used when normality assumptions were met; otherwise, the equivalent nonparametric test was used. Pearson\u2019s Chi-square test was used to analyze categorical variables. To compare the difference in topographic location between isolated and unisolated pontine infarction, we used Pearson\u2019s Chi-square test with post hoc analysis. P\u2009<\u20090.05 was considered statistically significant. SPSS 20.0 was used for statistical analysis.Continuous variables are expressed as the mean\u2009\u00b1\u2009standard deviation, and categorical variables are expressed as percentages. The differences in continuous variables were compared with the homogeneity test of variances. Student\u2019s P\u2009<\u20090.001).From May 1, 2003, to Oct 31, 2017, a total of 1140 patients with acute pontine infarction were screened , and 1003 patients were enrolled in the analysis  and tegmental infarction . Isolated pontine infarction had more anteromedial pontine infarction , although it was the most common subtype for both isolated and unisolated pontine infarction. Anterolateral and bilateral pontine infarctions were similar between isolated and unisolated pontine infarctions.As shown in Table P\u2009=\u20090.002, Table P\u2009=\u20090.000, Table The mechanism of pontine infarction was classified into LAD, BABD and LAD. For isolated pontine infarction, BABD was the leading cause  pontine infarction (including isolated and unisolated pontine infarctions) was mostly located in the ventral pontine, which was divided into anteromedial and anterolateral; (2) compared to isolated pontine infarction, unisolated pontine infarction had more unilateral multiple and tegmental infarctions but fewer ventral medial pontine infarctions; (3) most isolated pontine infarctions were caused by BABD, while most unisolated pontine infarctions were caused by SAD. At present, our study has the largest number of patients with acute pontine infarction.Based on our data, pontine infarction  was mostly located in the ventral pontine. Silverstein et al. found that 51% of infarctions were located in the ventral pontine in 81 autopsied cases. These autopsied cases may represent severe pontine infarction, which may result in selection bias. For isolated pontine infarction, Claudio et al. found that 58% of isolated pontine infarctions were located in the ventral pontine in an MRI-based study [For the tegmental pontine, Claudio et al. showed that 31% of isolated pontine infarctions were located in the tegmental pontine , while EThe topographic location indicated the possible mechanism of pontine infarction. This was proved by the previous studies , 3 and oLimitations First, this study was MRI based, and some patients who could not receive MRI scanning, such as those with pacemakers, were not enrolled. Second, the mechanism of pontine infarction was mostly diagnosed based on the topographic location, which might have caused some bias. Finally, we found that the mechanism of pontine infarction was related to the topographic location. However, the role of topographic location in prognosis remains unknown.This large series study of pontine infarction revealed the topographic location of unisolated pontine infarction."}
+{"text": "Lactobacillus rhamnosus GG (LGG) are known to adhere to milk components, which may impact their distribution and protection within dairy matrices and therefore is likely to modulate the efficiency of their delivery. However, the adhesive behavior of most LAB, as well as its effect on food structuration and on the final bacterial distribution within the food matrix remain very poorly studied. Using a recently developed high-throughput approach, we have screened a collection of 73 LAB strains for their adhesive behavior toward the major whey protein \u03b2-lactoglobulin. Adhesion was then studied by genomics in relation to common bacterial surface characteristics such as pili and adhesion-related domain containing proteins. Representative adhesive and non-adhesive strains have been studied in further depth through biophysical measurement using atomic force microscopy (AFM) and a relation with bacterial distribution in whey protein isolate (WPI) solution has been established. AFM measurements have revealed that bacterial adhesion to \u03b2-lactoglobulin is highly specific and cannot be predicted accurately using only genomic information. Non-adhesive strains were found to remain homogeneously distributed in solution whereas adhesive strains gathered in flocs. These findings show that several LAB strains are able to adhere to \u03b2-lactoglobulin, whereas this had only been previously observed on LGG. We also show that these adhesive interactions present similar characteristics and are likely to impact bacterial location and distribution in dairy matrices containing \u03b2-lactoglobulin. This may help with designing more efficient dairy food matrices for optimized LAB delivery.In the last decade, there has been an increasing interest in the potential health effects associated with the consumption of lactic acid bacteria (LAB) in foods. Some of these bacteria such as  Adhesion is a major property of microorganisms which effectively impacts microorganism activities as well as human health, and has been identified as a key factor involved in microorganism ecology. Adhesion enables bacteria to stick to both biotic and abiotic surfaces. Adhesion to abiotic surfaces leads to biofilm formation, which has been widely studied in relation to the food industry . AdhesioBacteria have also been shown to be able to adhere to food components, especially to meat  and moreLactobacillus rhamnosus GG (LGG) and \u03b2-lactoglobulin is mediated by the pili produced by LGG cells on their surface  after the addition of MRS culture growing medium  in the wspaCBA CMPG 5357 impaired in pili synthesis, which adhesive properties of both are well-known .For each series of experiments, a 96-well microplate previously stored at \u221280\u00b0C was thawed and replicated on working microplates using 50 \u03bcL of bacterial suspension to inoculate 150 \u03bcL of MRS by well. The working microplates were incubated at 30\u00b0C 2 days before the adhesion assay. During the adhesion assay, microplates were only centrifuged once at 1,642 \u00d7 Beta-lactoglobulin  was prepared in solution (1% w/w) as described by nm595  measurements over 48 h.Adhesion and growth monitoring were done according to start) were monitored such as described by tstart (highest adhesion) obtained on a control without \u03b2-lactoglobulin and the highest tstart (lowest adhesion) obtained on \u03b2-lactoglobulin:The times at which the apparent bacterial growth starts  for normal data and Wilcoxon\u2013Mann Whitney and Steel-Dwass tests (non-parametric) for data that did not fit normal distribution using Kyplot software (Kyens Lab Inc.).Statistical analysis were performed via Protocols used in this part have been adapted from Lactobacillus aquaticus DSM 21051 and Lactobacillus sharpeae DSM 20505 were prepared by inoculating 9 mL of MRS broth with 100 \u03bcL of bacterial stock and grown overnight at 37\u00b0C. These precultures were used to inoculate 9 mL of fresh MRS broth the next day and the growth was performed at 37\u00b0C until an optical density of 1.2 was reached at 660 nm (for about 8 h). Cultures were then centrifuged at 3,000 \u00d7 g for 10 min at room temperature. Pellets were suspended in 1 mL of PBS (pH 6.8).Cultures were prepared according to 2-terminated PEG-linker  was used, as well as AFM probes with borosilicate glass particle (2 \u03bcm), coated with gold and modified with NH2 terminated PEG linker . The bacterial suspension is deposed on mica at 4\u00b0C and left overnight (pH 6.8). Preparation of the \u03b2-lactoglobulin and Bovine Serum Albumine (BSA) 1% (w/w) solutions  was done according to According to \u22121. Force distance curves were recorded between the bacteria deposited on functionalized mica and the probe coated with \u03b2-lactoglobulin or BSA. Three adhesion force maps  were recorded for each protein-bacteria interaction analysis. Data analysis was performed using the Nanoscope Analysis software from Bruker  and the last peak was calculated for each curve before plotting adhesion forces and last rupture length histograms. The last peak is used for analysis instead of the maximum peak in order to characterize the last interacting point between the \u03b2-lactoglobulin and the cell receptor and not the unfolding of a biomolecular domain.Protocol followed is described by g for 10 min at room temperature. Pellets were suspended in 10 mL of WPI solution . The WPI solution was prepared using PRODIET 90 S  that is a soluble milk protein isolate containing native whey proteins including \u03b2-lactoglobulin. One milliliter of resuspended cells was stained with the LIVE/DEAD BacLight viability kit . Two hundred microliters of LAB suspension  were introduced on chambered glass slides . CLSM images were taken using a Leica TCS SP5-X-AOBS confocal laser scanning microscope  equipped with WLL lasers. The objective lens used was a HCX PL APO CS 100 \u00d7 1.40 (oil immersion). The excitation wavelength was 488 nm and emission bandwidth was of 495\u2013510 nm for SYTO 9 and 600\u2013620 nm for propidium iodide. Two independent repetitions were performed and approximately 20 representative images were acquired for each sample.The cultures were prepared as described in Section \u201cBacterial Cultures\u201d, then centrifuged at 3,000 \u00d7 spaCBA (\u2212386), known to be non-adhesive to \u03b2-lactoglobulin (L. aquaticus DSM 21051 (MAV = 61.5), Lactobacillus murinus DSM 20452 (MAV = 12.8), Lactobacillus plantarum DSM 13273 (MAV = 12.6), Lactobacillus brantae DSM 23927 (MAV = 6.97), although these strains were still less adhesive than the positive control LGG WT (MAV = 104). Nine strains were also found to have a MAV inferior to the one of the negative control LGG spaCBA: Lactobacillus sharpeae DSM 20505 (MAV = \u2212857), Lactobacillus kefiri DSM 20587 (MAV = \u2212787), Lactobacillus similis DSM 23365 (MAV = \u2212780), Lactobacillus pobuzihii DSM 28122 (MAV = \u2212617), Lactobacillus namurensis DSM 19117 (MAV = 516), Lactobacillus satsumensis DSM 16230 (MAV = \u2212490), Pediococcus parvulus DSM 20332 (MAV = \u2212477), Lactobacillus senmazukei DSM 21775 (MAV = \u2212404), Lactobacillus lindneri DSM 20690 (MAV = \u2212387). The MAV for all strains are listed as Supplementary Data Most strains were found not to be adhesive to \u03b2-lactoglobulin as the average MAV calculated on the 73 strains was negative (\u2212180 \u00b1 22) although higher than the MAV of the negative control LGG globulin . The micL. aquaticus DSM 21051 (the most adhesive strain) and L. sharpeae DSM 20505 (the least adhesive strain) were studied through AFM, in order to characterize them in further depth. Only two strains were chosen to precise our understanding of the interaction mechanism of the LAB surface with \u03b2-lactoglobulin since AFM is not a suitable method for screening of large populations. This is why we decided to select only the two strains at the extreme of the adhesion spectrum for this analysis. BSA was used as a negative control as LAB strains have previously been found to feature low adhesion to it  and are not repeated within a given protein.More predicted surface characteristics were analyzed for the four strains found to be adhesive to \u03b2-lactoglobulin. Predicted protein domains featuring LPxTG motif found for each strain are listed in r LGG WT  but no hr LGG WT . Other aL. aquaticus DSM 21051, the most adhesive strain to \u03b2-lactoglobulin. MucBP domains have been found predominantly in lactobacilli found naturally in intestinal niches, which suggests that they play an important role in establishing host-microbial interactions in the gut by binding mucus (L. plantarum DSM 13273 is the strain featuring the highest number of adhesion-related domains in its genome (L. brantae DSM 23927 features leucine-rich repeats (LRRs) and SD-repeat (Sdr) domains (Staphylococcus aureus adhesion and pathogenesis (L. brantae DSM 23927. No other adhesion-related domain than the Ig-like fold domain was identified on L. murinus DSM 20452 (The MucBP domain is the only domain with a known adhesive-related function (apart from the Ig-like fold domain) which could be identified on ng mucus . L. plans genome . This iss genome  and ther domains , both of domains  and are  domains . Sdr-repogenesis . The proSM 20452 , which wThe aim of this study was to evaluate and characterize adhesive interactions occurring between LAB and \u03b2-lactoglobulin. A collection of 73 LAB strains was screened for their adhesive behavior toward \u03b2-lactoglobulin and strains at the extreme of the adhesion spectrum i.e., a highly adhesive and a poorly adhesive strains were studied in further depth.Only four strains out of 73 were found to present adhesive affinities toward \u03b2-lactoglobulin. Therefore, adhesion to \u03b2-lactoglobulin appears not to be a common characteristic of the LAB group. The consequences of these adhesive interactions, when they occur, are not fully understood. However, it could be hypothesized that strains featuring adhesive affinities toward whey proteins would be lost during the drainage step of cheese manufacturing processes, alongside with whey expulsion from the cheese network. It would be interesting to test the affinity of this same strain collection to other food components in future work, in order to dispose of more comparison points to our study and to get a better understanding of the importance of adhesion to \u03b2-lactoglobulin compared to adhesion to other food components. Currently, the rare existing studies discussing bacterial adhesion to food components other than \u03b2-lactoglobulin concern up to four strains at most at a time , therefoL. lactis. Out of 55, 30\u201340 strains presented adhesive affinities toward casein-derived components, depending on their growth phase, and strains isolated from a dairy environment presented much stronger binding of milk proteins versus strains isolated from plants, suggesting a selective advantage .The study performed by dvantage . HoweverL. aquaticus DSM 21051, exhibited a specific adhesive behavior when studied by AFM. The signature of the observed retraction curves was identified as specific of biomolecules stretching, suggesting that the surface of L. aquaticus DSM 21051 features a strong affinity toward \u03b2-lac. This has also been shown previously for the model strain LGG WT by our team as well as for the mutant strain LGG welE, expolysaccharide-depleted and known to adhere more to \u03b2-lactoglobulin than LGG WT due to its increased pili exposure . Similarly, our team demonstrated previously this same fact for the model strain non-adhesive to \u03b2-lactoglobulin, LGG spaCBA . Overall, L. sharpeae DSM 20505 has demonstrated very poor adhesive capacities toward \u03b2-lactoglobulin. However, when analyzed for predicted adhesion-related protein domains, this strain revealed a total of 23 adhesion-related domains, 8 of which being different, including MucBP and gram-positive pilin subunit D1 N-terminal, although no sequence homologue to the spaCBA domain was found (data not shown). The spaCBA domain is known to mediate adhesion to \u03b2-lactoglobulin for the piliated strain LGG WT  which would impact host colonization, but may also better protect bacterial survival until they reach the GIT. These findings also pave the road to future experiments aiming generalizing bacterial adhesion characteristics to broad bacterial groups, thus helping with practical food matrix design. It would therefore be interesting to study the potential protective effect of components to which bacteria are adherent during critical steps of the food manufacturing process, such as spray-drying during probiotic milk powder production.FG, JG, JB, FB, and CG conceived the study. FG, JG, JB, SE-K-C, DD, and GF carried out the experiments. FG, JB, JG, SE-K-C, DD, and GF analyzed the data. FG, JG, and JB wrote the manuscript. All authors commented on 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."}
+{"text": "Although they share similar structural properties, we show that their electronic structure exhibit dramatic differences. A strong anisotropic Dirac dispersion is revealed in BaMnBi2 with a decreased asymmetry factor compared with other members of AMnBi2  family. In addition to the Dirac cones, multiple bands crossing the Fermi energy give rise to a complex Fermi surface topology for BaZnBi2. We further show that the strength of hybridization between Bi-p and Mn-d/Zn-s states is the main driver of the differences in electronic structure for these two related compounds.We investigate the electronic structure of BaMnBi Moreover, the linear crossing around the Fermi level (EF) is observed in broad categories of materials of strong contemporary interest, including topological insulators, d-wave superconductors, and iron-based compounds3.Dirac materials, characterized by the linear dispersion of their low-energy quasi-particle excitations, have received significant recent attention, given their potential to host various exotic phenomena such as high mobilities due to strongly suppressed backscattering, unconventional quantum Hall effects, and Klein tunneling5. The anisotropy enables the electrons to propagate differently depending on crystallographic direction, providing additional versatility for applications6. Significant effort in prior work has been undertaken to generate the anisotropy on Dirac cone using patterned superstructures, mechanical stress, and heterostructures, among others12.Anisotropic Dirac materials are distinguished by their strong momentum-dependent Fermi velocities on the Dirac cone2 , was reported to have intrinsic anisotropy in their Dirac dispersion. The role of the A-site cation on the Dirac dispersion has been studied extensively, where the dispersion has been shown to qualitatively change upon breaking mirror symmetry between Ca- and Sr-based AMnBi2 compounds13 or breaking time reversal symmetry inducing the Weyl semimetallic phase in Yb-based AMnBi2 compounds14. However, direct measurements of the electronic structure of the Ba-based AMnBi2 compound have yet to be reported; and BaMnBi2 would aid in furthering a systematic investigation of the relation between A-site ionic radii and the nature of the Dirac dispersion. More importantly, it has been shown by first-principles calculations that the partially filled Mn-d states do not directly contribute states near the EF13, unlike many other transition metal pnictides; yet the Mn atoms are known to possess net magnetic moments in the d5 high-spin state, possibly giving rise to an anomalous magnetoresistance15. Investigating the role of the transition metal d-states on low energy Dirac dispersion is challenging due to the multi-valence nature of these ions; the change in the orbital occupancy may induce a variety of ordered states and accompanying atomic distortions. To avoid such structural distortions from the d5 (Jahn-Teller inactive) configuration that lower symmetry, one must consider B-site ions with empty or fully filled d bands, such as AZnBi2, as we do below.Recently, a new family of materials, AMnBi2 and BaZnBi2 using angle-resolved photoemission spectroscopy (ARPES) and first-principles density functional theory (DFT) calculations. We find that using Ba on the A-site leads to anisotropic Dirac bands, although with different asymmetry and anisotropy from MnBi2 compounds. In addition, substantial changes in the electronic structure are found by substituting Zn with Mn , yielding more trivial bands at EF due to the increased hybridization between Zn and Bi states.In this report, we explore the electronic structure of BaMnBi2 and BaZnBi2. These materials consist of alternating layers of Bi-net and Mn (or Zn)-Bi tetrahedra separated by Ba layers located in mirror symmetric positions with respect to the Bi-net  and the other forming the Bi-tetrahedra around the Mn (or Zn) cations (defined as Bi2). It has been pointed out that states near EF of AMnBi2 compounds are dominated by Bi1-p orbitals whereas the Bi2-p states that hybridize with the Mn-d orbitals are away from the Fermi level13. We note that the c/a ratio of the BaZnBi2 is dramatically smaller than those reported for the Mn family, a reduction that can be primarily attributed to the reduced Zn-Bi distance and that reflects differences in the nature of bonding between Zn-Bi and Mn-Bi. With the small binding energy of the Bi2-p states in AMnBi213, the expected change in the bonding character in BaZnBi2 should induce a significant change in the states near the EF, which will be discussed later in our ARPES data and first-principles results.Figure\u00a02  of this class16. On the other hand, the BaZnBi2 FS shows new features, including double layers of large diamond-like pieces connecting the four X points in the Brillouin zone (BZ) and two concentric circle-like hole pockets around the \u0393 point. The four corners of the diamond-like FS features overlap with neighboring ones at the X points, generating additional small diamond-like electron pockets at these four X points in the BZ.Fermi surfaces (FSs) of BaMnBi2 Fig.\u00a0 and BaZn.\u00a02 Fig.\u00a0 measured2 and BaZnBi2 from our ARPES measurements are illustrated on Figs\u00a02 at different binding energies are shown in Fig.\u00a0B\u2009=\u2009100\u2009meV. Our first-principles DFT calculations within the generalized gradient approximation (GGA) and including spin-orbit (SO) interactions, shown with black solid lines on Fig.\u00a0Further investigations of the electronic structures of BaMnBivL) and the right (vR) branches are ~6\u2009eV\u00b7\u00c5 and ~1.9\u2009eV\u00b7\u00c5, respectively, which yields an asymmetry value ((vL\u2009\u2212\u2009vR)/vL) ~0.68. This is smaller than the asymmetry values of CaMnBi2 (0.80) and SrMnBi2 (0.78)16. It is well known that this asymmetry originates from the strong spin-orbit coupling which also gaps out the Dirac band16. The doubly-degenerate Dirac cones associated with the low energy electronic structure along \u0393-M and absence of states along \u0393-X direction is consistent with our first-principles calculations, which will be discussed in more detail below.To analyze these anisotropic Dirac bands further, we examine the measured electronic band dispersion along four cuts from A to D Fig.\u00a0. The Dirv\u2225\u2009=\u2009(vL+vR)/2) and perpendicular to \u0393-M (v\u22a5), which yield ~4\u2009eV\u00b7\u00c5 and ~0.3\u2009eV\u00b7\u00c5, respectively. The anisotropy value can be defined as v\u2225/v\u22a5\u2009~\u200913, which is smaller than CaMnBi2 (64), YbMnBi2 (209), but roughly equivalent to SrMnBi2 (13)16. This illustrates a correlation of the strength of the anisotropy with space group, since CaMnBi2 and YbMnBi2 with P4/nmm display relatively stronger anisotropy than SrMnBi2 and BaMnBi2 that belong to I4/mmm.The crossing points of Dirac bands are the highest in energy along \u0393-M directions and shift downwards with momentum away from the \u0393-M directions (from cut A to cut D). At the same time, on cut C and D, the Dirac band dissected along the perpendicular direction compared to the cut A appears around the \u0393 point. The slope of the \u0393-point Dirac band is much less steep compared to the one in cut A, which clearly exhibits the strong anisotropic characteristics expected from a Dirac band. For a quantitative analysis of the anisotropy of Dirac bands, we extract the Fermi velocity along \u0393-M .We substitute Mn with Zn to investigate the effect of their different valence configurations on the band structure and anisotropy. The constant energy contours measured for BaZnBi2 Fig.\u00a0 display .\u00a02 Fig.\u00a0 with two2 and BaZnBi2 along high symmetry lines are shown in Fig.\u00a02 and exhibit degenerate Dirac cones along the \u0393-M direction and absence of the states along the \u0393-X direction, consistent with the experimental data around the Fermi level. The steep dispersion at the Dirac cone originates with the Bi-p orbitals arranged in the square net, as shown from our calculated projected band dispersions  of BaMnBisee Fig.\u00a0. In BaMnd-p vs. s-p hybridization), we compare the band structures of BaMn(Zn)Bi2 calculated with relaxed atomic structure of BaMn(Zn)Bi2 and with deliberate changes of Mn(Zn) \u2013 Bi distance for that of BaZn(Mn)Bi2 in Fig.\u00a0EF, it would be ideal to choose transition metal ions with frontier orbitals that hybridize weakly with the Bi2-p orbitals and that are gapped at the Fermi level.In order to distinguish between the effect of the change in structure (Mn/Zn to Bi distance) and the change in bonding character  parametrization20 are used for the GGA exchange correlation functional. Spin-orbit coupling is included self-consistently for all the calculations. We use the projector augmented wave method21 with an energy cutoff of 500\u2009eV and k-point sampling on a 6\u2009\u00d7\u20096\u2009\u00d7\u20092 grid. The atomic positions are fully relaxed until Hellmann-Feynman forces are less than 0.02\u2009eV/\u00c5. The lattice constants relaxed with the PBE functional are in good agreement with experiment for BaMnBi2 with difference of 1.5% for volume and \u22120.04% for c/a ratio. For BaZnBi2 the unit-cell volume calculated by the GGA shows reasonable agreement with 2.3% error with respect to the experimental value but the c/a ratio deviates more significantly, with an error about 8.5% compared with experimental value. The deviation in the c/a ratio does not change significantly with the inclusion of the long-range Coulomb interaction using hybrid functional22 but decreases with van der Waals interaction (vdW-D3)23 to 5%. Since there are only small changes in the band structures by including vdW, the band structures using GGA exchange correlation functional are presented both BaMnBi2 and BaZnBi2. The band structures of BaMnBi2 are calculated with checkerboard type of antiferromagnetic ordering for the Mn-d spins; the checkerboard order is calculated to have the lowest total energy which is 0.08 and 0.26\u2009eV per formula unit lower than stripe-type antiferromagnetic ordering and ferromagnetic ordering, respectively, consistent with other AMnBi2 compounds24. Fermi surfaces are calculated by interpolating energy dispersion using dense k-grid points (80\u2009\u00d7\u200980) at kz\u2009=\u20090.We perform first-principles density functional theory calculations with the generalized gradient approximation (GGA) method using the Vienna"}
+{"text": "Nowadays, stress is considered one of the most significant health problems in modern society, related to the physiopathology of psychiatric, metabolic and cardiovascular diseases, and the search for its biomarkers remains a challenging task for researchers and clinicians.The association between mental stress and cardiac function has always been of major interest. In the 19But what is stress? How can we define it, before we tackle it?From a phylogenetic point of view, the perception of threat and safety is the core element implicated in mental events related to stress. This threat appraisal works as a trigger of complex neurological processes leading to adaptive adjustments of heart rate, cardiac contractility and vascular resistance that follows sympathetic tone enhancement and parasympathetic withdrawal, and ultimately result in the survival of the individual and of the species. In summary, the heart and the brain are in constant communication in order to keep us from danger.1 identified an increase in the low frequency spectral power and in total spectral inequalities (using the Gini coefficient) during a cognitive mental challenge (arithmetic exercise). Interestingly, the 0.1Hz band expression, frequently associated with the arterial baroreflex activation, was significantly increased. Authors therefore proposed these indexes as biomarkers of stress and implicated baroreflex hyperactivity in its psychophysiology.Although several physiological mechanisms are known to take part in this elaborate neurological circuitry, the autonomic nervous system is, undisputedly, the protagonist. The study \"Introduction of Application of Gini Coefficient to Heart Rate Variability Spectrum for Mental Stress Evaluation\",To the extent that we assume autonomic tonus changes as a reliable sign of mental stress, the heart rate variability (HRV) may indeed provide some interesting information. However, a careful appreciation of the involved neural structures may yield an insight into a sophisticated system and indicate that a reductionist interpretation of the autonomic response pattern may turn out to be misleading.2 Close to the epicardial surface, this innervation resolves into the intrinsic system, consisting of a dense net of thousands of neural cells and hundreds of epicardial ganglia, plentifully located in the atrial surface.4 Cardiac ganglia work as integrative centers, where efferent data can be modulated, so the whole system can flexibly respond to a wide range of stimuli.5 This modulation, this capacity to provide adaptive control over the periphery, is the hallmark of the autonomic nervous system.Observe the peripheric autonomic nervous pathways carefully. Its extrinsic components, mainly comprised by the vagus nerve and the numerous afferent and efferent nerves of the sophisticated sympathetic thoracic chain, are responsible to carry, through the central nervous system and back to the heart, sympathetic and parasympathetic information from baro and chemoreceptors, generating balanced responses that maintain internal homeostasis. Take into consideration that most of these structures are bimodal, with both vagal and sympathetic inputs.7 describe a central autonomic network,7 containing cortical and subcortical areas, through which the brain controls visceromotor functions and goal-directed behavior. The network includes prefrontal cortices, the central nucleus of the amygdala, the paraventricular nucleus of the hypothalamus, the parabrachial nucleus, the nucleus of the solitary tract, and the nucleus ambiguous, among others. All these components are reciprocally interconnected, and the interplay of these inputs provides flexible adjustments. The system essentially operates as a continuous integration of concepts such as \u201cself\u201d and \u201cdanger\u201d with external perceptions and memory into Gestalt representations, generating likely responses.Intrinsic and extrinsic systems are connected to the central nervous system. Here is where things start getting tricky. By using PET Scan and MRI, a series of neuroimaging studies7 The more abstract the stressful event, the more important and modulated the inhibition of subcortical cardioacceleratory circuits are, meaning that all these neural structures can be differentially recruited depending on the nature of the challenge, creating context-specific response patterns.After appraising a potential threat, a primitive quick mental stress reaction arises from the amygdala. The reaction to uncertainty or danger is a relatively simple sympathoexcitatory state known as \u201cfight or flight\u201d, that in its pristine form results in a rather predictable HR increment. However, this initial perception often gives way to more elaborate mental interpretations as certain cortical areas action unfolds. The frontal cortex (FC), and medial preFC in particular, has a significant role by activating GABAergic pathways exerting inhibitory control over an activated amygdala.8 The resultant HRV takes so many variables into account that defining specific spectral bands as reliable biomarkers, assuming a mechanistic causality, seems unlikely, especially so when it comes to abstract stressful contexts, such as expectations of future outcomes, emotional conditions and representation of economic values.7Complex tasks requiring cognitive functions, such as arithmetic, online processing and manipulation of information are highly dependent on this reciprocal downregulation between the cortex and the amygdala.7To illustrate this limitation, in a very simplified way, imagine you are about to take a math test. At first, the challenge may disturb you, being felt as \u201cdanger\u201d, and so the amygdala nucleus promptly triggers a vagal withdraw  and a sympathoexcitatory reflex (modifying low frequency spectra). A few minutes later you realize you can handle it and start feeling confident. It is just going to take a little focus. A more accurate Gestalt representation has now been reached. By activating a GABA pathway, three main different areas of your preFC engage: 1-posterior and dorsal region of the rostral preFC (linked to cognitive functions), 2-dorso medial preFC  and finally 3- medial-orbitoFC and anterior ventral preFC .7Exerting a balanced inhibitory control over the amygdala through an integrated Central Autonomic Network, these anatomic structures guide goal-directed behavior and adaptability and ultimately dictates the amount of acetylcholine and norepinephrine to be released from the post-ganglionic fibers close to the sinus node, reshaping HRV spectra once again. Keep in mind that genetic background and cognitive performance may influence all these processes significantly.9 had already identified inter-beat interval patterns preceding severe fetal distress even before a perceivable change in fetal heart rate. Undoubtedly, HRV has been proven to be an essential index of adaptability of the organism, and therefore, extensively studied under a wide range of stressful stimuli. Conflicting results, however, suggest distinct reactions to different forms of stress. While some authors demonstrated a global increase in HRV after exposure to noise,11 public speech tasks12 and sustained attention,13 others found a global HRV reduction during memory14 and cognitive tasks.15 Emotional conditions have been proven to correlate with high-frequency band reduction by some authors,16 and yet to be neutral by others.12What is the value of HRV on assessing stress, then? As of 1965, Hon and LeeWe are far from relying on surrogate endpoints to understand the different parts of the link between heart and brain. Even by using much more sophisticated techniques, such as regional cerebral blood flow neuroimaging, we are still scratching the surface of this intricate physiology. HRV could be an interesting tool for detecting general features of the stress response, although unreliable for distinguishing its complex mechanisms."}
+{"text": "However, the regulation of ANO1 surface expression in glioblastoma cells is largely unknown. In this study, we found that Ca2+/Calmodulin-dependent protein kinase II (CaMKII) \u03b2 specifically enhances the surface expression and channel activity of ANO1 in U251 glioblastoma cells. When KN-93, a CaMKII inhibitor, was used to treat U251 cells, the surface expression and channel activity of ANO1 were significantly reduced. Only CaMKII\u03b2, among the four CaMKII isoforms, increased the surface expression and channel activity of ANO1 in a heterologous expression system. Additionally, gene silencing of CaMKII\u03b2 suppressed the surface expression and channel activity of ANO1 in U251 cells. Moreover, gene silencing of CaMKII\u03b2 or ANO1 prominently reduced the migration and invasion of U251 and U87 MG glioblastoma cells. We thus conclude that CaMKII\u03b2 plays a specific role in the surface expression of ANO1 and in the ANO1-mediated tumorigenic properties of glioblastoma cells, such as migration and invasion.ANO1, a Ca The m channel , and Ca2channels . Recentlchannels ,11.2+-activated Cl\u2212 channel (CaCC) that is activated by intracellular Ca2+ [ANO1 was identified as a Calar Ca2+ ,13,14 anlar Ca2+ ,16,17. Alar Ca2+ . In addilar Ca2+ ,20,21,22lar Ca2+ ,24. Base2+/Calmodulin-dependent kinase II (CAMKII) signaling in breast cancer cells [Several recent studies have shown that ANO1 is involved in oncogenic signaling. In HNSCC, ANO1 contributes to the activation of mitogen-activated protein kinase (MAPK) and protein kinase B  by interacting with EGFR . The acter cells . In addier cells . We alsoer cells ,11. Alth2+/Calmodulin complex [\u03b3 inhibits native CaCC currents, and the serine 727 mutant (S727A) of ANO1 reverses the CaMKII\u03b3-mediated inhibition of ANO1-mediated currents [The CaMKII family of proteins are multifunctional serine/threonine-specific protein kinases that are regulated by the Ca complex . The CaM complex . CaMKII  complex . These C complex ,27,28. Icurrents . Anothercurrents . Despitecurrents , the effIn the present study, we found that surface expression and channel activity of ANO1 are enhanced in a CaMKII\u03b2-specific manner in U251 and U87 MG glioblastoma cells. We also found that suppression of CaMKII\u03b2 using small interfering RNA (siRNA) or a CaMKII inhibitor, KN-93, reduced the surface expression and channel activity of ANO1 in glioblastoma cells. In addition, specific gene silencing of CaMKII\u03b2 and/or ANO1 suppressed the migration and invasion of glioblastoma cells. Our results provide strong evidence for a CaMKII\u03b2-dependent regulation mechanism of ANO1 surface expression and indicate potential therapeutic targets against ANO1-mediated tumorigenesis in glioblastoma cells.4,4\u2032-Diisothiocyano-2,2\u2032-stilbenedisulfonic acid (DIDS), a broad-spectrum chloride channel blocker was purchased from Tocris . CaCCinh-A01, an ANO1 inhibitor, and KN-93, a CaMKII inhibitor were purchased from Sigma-Aldrich . 4\u2032,6-diamidino-2-phenylindole (DAPI) staining solutions was purchased from Thermo Fisher Scientific . Other reagents were purchased from Sigma-Aldrich.2 -containing atmosphere. Cells were seeded on poly-L-lysine (10 \u03bcg/mL) pre-coated plates and then expression vectors for CaMKII isoforms were transfected into cells using jet PEI  according to the manufacturer\u2019s protocol. To silence CaMKII\u03b2 expression, CaMKII\u03b2 small-interfering RNA (CaMKII\u03b2 siRNA)  was transfected using INTERFERin\u00ae siRNA transfection reagent , according to the manufacturer\u2019s protocol.U251 and U87 MG glioblastoma cell lines were cultured in Dulbecco\u2019s modified Eagle\u2019s medium (DMEM)  containing 10% fetal bovine serum (FBS)  and 100 units/mL penicillin-streptomycin . Cultures were maintained at 37 \u00b0C in a humidified, 5% CO5 cells/dish) for 24 h and then infected with lentivirus in the presence of polybrene (8 \u00b5g/mL) for 72 h.To produce lentivirus containing specific short hairpin-forming RNA against ANO1 (ANO1 shRNA), we purchased the TMEM16 (ANO1) Human shRNA Plasmid Kit from OriGene , which comprises a control lentiviral scrambled shRNA vector (Sc shRNA) and four individual lentiviral vectors encoding ANO1 shRNAs (shANO1A~shANO1D). Among the four lentiviral vectors encoding ANO1 shRNAs, we selected the most efficient lentiviral vector containing shANO1D. Sequences of shANO1D is as follows: 5\u2032-GCTGAATACGAAGCCAGAGTCTTGGAGAA-3\u2032. Using the control lentiviral vector encoding Sc shRNA and lentiviral vectors encoding ANO1 shRNA, lentivirus production was performed according to the protocol described in a previous report . Viral pTo construct expression vectors, we routinely used the Gateway recombination cloning system (Invitrogen). The BP clones encoding human CaMKII\u03b1 , human CaMKII\u03b2 , human CaMKII\u03b4 , and human CaMKII\u03b3  were purchased from Addgene. These genes were transferred from BP clones to several destination expression vectors, such as pDEST-HA-N, pDEST-Flag-N, and pDEST-mCherry-N by the Gateway LR recombination reaction (Invitrogen), according to the manufacturer\u2019s guidelines.\u2212\u2206\u2206CT method was used to calculate fold changes in gene expression. All experiments were repeated at least three times.Total RNA was isolated from U251 cells or U87 MG cells using an RNA purification Kit , according to the manufacturer\u2019s instructions. RT was performed with 1\u2009\u03bcg total RNA using a cDNA Synthesis Kit , according to the manufacturer\u2019s instructions. PCR was performed using Pfu Plus 5x PCR premix  under the following cycle conditions: Denaturation at 95 \u00b0C for 20 s, annealing at 55 \u00b0C for 20 s, and extension at 72 \u00b0C for 30 s. This cycle was repeated a total of 30 times. The PCR products were separated by electrophoresis in a 2% agarose gel, and images were captured on a gel imaging system. qPCR was also performed with SYBR Green mix . \u00ae 647 conjugate (WGA647)  at 4 \u00b0C for 15 min to label the plasma membrane. Cells were then permeabilized with Triton X-100 (0.5% in PBS) and blocked with 5% bovine serum albumin for 1 h at room temperature. Subsequently, the cells were incubated overnight at 4 \u00b0C with anti-ANO1  antibody; after washing, cells were incubated with DyLight 488-conjugated secondary antibody  for 1 h at room temperature. The cells were then washed three times, mounted after drying, and observed under a Nikon A1 confocal microscope.U251 cells growing on coverslips were incubated with KN-93 (10 \u03bcM); they were then transfected with CaMKII\u03b2 siRNA to silence the expression of CaMKII\u03b2 and incubated for an additional 24 h. Cells were fixed in 4% paraformaldehyde (PFA) for 20 min at room temperature (about 20\u201325 \u00b0C) and then incubated with Wheat Germ Agglutinin, Alexa FluorFor the surface biotinylation assay, U251 cells transfected with CaMKII\u03b2 siRNA or CaMKII isoforms were incubated at 4 \u00b0C and washed with PBS twice. Plasma membrane proteins were then biotinylated in PBS containing sulfo-NHS-LS-biotin  for 30 min. After biotinylation, cells were washed with quenching buffer (100 mM glycine in PBS) and then washed with PBS two times. Cells were then lysed and incubated with high-capacity NeutrAvidin-agarose resin . After three washes with lysis buffer, bound proteins were eluted in SDS sample buffer and separated using 10% SDS PAGE electrophoresis; they were then transferred to PVDF membranes. After blocking, membranes were incubated overnight at 4 \u00b0C with anti-ANO1 , anti-Flag , or anti CaMKII\u03b2  antibody and were then incubated with HRP-conjugated anti-mouse, anti-rat, or anti-rabbit IgG antibody.I\u2013V) curves were obtained using U251 cells transfected with mCherry-CaMKII isoforms, and whole-cell currents were measured by applying 1-s duration voltage ramps from +100 to \u2212100 mV  at room temperature. For measuring Cl\u2212 currents, recording electrodes (4\u20137 M\u03a9) were filled with (mM): 146 CsCl, 8 HEPES, 5 Ca-EGTA-NMDG, 2 MgCl2, and 10 sucrose (pH adjusted to 7.3 with CsOH). The standard bath solution contained (in mM) 139 NaCl, 3 KCl, 10 HEPES, 2 MgCl2, 2 CaCl2, and 5.5 glucose (pH adjusted to 7.3 with NaOH). Whole-cell currents were amplified using the Axopatch 200A patch clamp system. Acquired data were analyzed using the pCLAMP 10.2 software (Molecular Devices). Current-voltage  or 24 h (U87MG cells). Phase contrast images were captured using ECLIPSE Ts2 inverted Routine Microscope . Cancer cells were rapidly grown in a small space of SPLScar\u2122 Scratcher culture dish, phase of the growing cells can be different from that of initially seeding cells. Analyses were performed using image J which select outlines where the wound is partially closed. The ratio of the remaining wound area was calculated relative to the initial wound area and normalized to that for scrambled shRNA-infected cells and scrambled siRNA-transfected cells. The error bars in graphs denote the standard error of the mean (s.e.m). All experiments were performed in triplicate.Glioblastoma cells were infected or transfected with ANO1 shRNA lentivirus or CaMKII\u03b2 siRNA. These cells were plated onto SPLScar\u2122 Scratcher culture dish  at a density of 1 \u00d7 105 cells/well in 100 \u03bcL DMEM. The lower chambers were filled with 500 \u03bcL DMEM. The trans-wells were incubated for 18 h to allow cell migration. Following incubation, the cells from the upper side of the insert filter were completely removed using a cotton swab, and those that had invaded through the coated membrane to its lower surface were fixed with 100% ethanol and stained with hematoxylin and eosin. For quantification, cells were counted under a microscope in three random fields at 20\u00d7 magnification. All experiments were performed in triplicate.Trans-well invasion chambers with 8.0 mm pores  were used, according to the manufacturer\u2019s instructions. Growth factor reduced Matrigel  was used to coat the membrane for 5 h. U251 cells or U87 MG cells infected with ANO1 shRNA lentivirus or transfected with CaMKII\u03b2 siRNA were seeded onto the transwell membrane insert at a density of 1 \u00d7 103 cells/well and incubated for 24 h. Cells were infected or transfected with ANO1 shRNA lentivirus or CaMKII\u03b2 siRNA. After 48 h, the reagents of 3--2,5-diphenyl tetrazolium bromide (MTT) was made with D-Plus\u2122 cell counting kit (CCK) cell viability assay kit  according to the manufacturer\u2019s protocol. These reagents were added to each well and the cells were incubated for 1 h at 37 \u00b0C. After incubation, Dimethyl sulfoxide (DMSO) is added at the end of the reaction after the medium from cells were removed to dissolve the formazan crystals formed from the reaction. After 30 min, the absorbance was measured at 450 nm wavelength using a spectrophotometer .U251 cells were seeded in 96-well plates at a density of 5 \u00d7 10p < 0.05, ** p < 0.01, or *** p < 0.001).Statistical analysis was performed using Origin software . The sample size employed was based on previous studies and was not predetermined by a statistical method. No randomization method was used. Data distribution was assumed to be normal, but this was not formally tested. Numerical data are presented as mean \u00b1 standard error of the mean (s.e.m). The variances were similar between the groups compared. Statistical significance was assessed using an unpaired or paired Student\u2019s t-test, with the significance level denoted by asterisks (* I\u2013V) relationship of chloride currents in U251 cells showed outward and inward currents  (p = 0.014)  A,B. ANO1= 0.014) C,D. We anhibitor E,F. Figup = 0.0005)  . These dWe examined the effects of CaMKII\u03b2 knockdown on the surface expression and channel activity of endogenous ANO1 in U251 cells using CaMKII\u03b2 siRNA. The knockdown efficiency of CaMKII\u03b2 siRNA was examined by qPCR and Western blotting experiments A,B. The p = 0.009)  D,E. In a= 0.009) F,G.These data clearly demonstrated that gene silencing of CaMKII\u03b2 reduces the surface expression of ANO1, as well as ANO1-mediated chloride currents in U251 glioblastoma cells.We recently reported that surface expression and channel activity of ANO1 are critical in the tumorigenesis of U251 cells . Since AAs expected, depletion of ANO1 or CaMKII\u03b2 in U251 cells resulted in a significant decrease in the invasiveness (as assessed by the collagen-coated transwell invasion assay) of U251 cells compared to the invasiveness of Sc shRNA-and Sc siRNA-treated cells C,D. In aFinally, we examined whether the deficiency of CaMKII\u03b2 or ANO1 also affects other glioblastoma cells. We tested the roles of CaMKII\u03b2 and ANO1 in the tumorigenesis of U87 MG cells which is another glioblastoma cell showing high expression of ANO1 . As showCumulatively, these results strongly suggest that suppression of ANO1 surface expression by knockdown of CaMKII\u03b2 is critical in the progression of human glioblastoma.We previously reported that the surface expression and channel activity of ANO1 are critical for the tumorigenesis of glioblastoma cells . HoweverCaMKII has been identified as a factor in the proliferation, migration, and survival of various cancer cells, such as those of lung, breast, prostate, and colon cancers ,34,35,362+ concentration is critical for the cancerous progression of glioblastoma cells [2+ [2+ can enhance surface expression and channel activity of ANO1 via activation of CaMKII\u03b2. The relationship between receptor-mediated Ca2+ signaling and ANO1-mediated tumorigenesis should be investigated in a future study. It is noteworthy that previous studies demonstrated functional interactions between CaMKII and ANO1 chloride channels ,29,30. Ima cells ,39,40. Sells [2+ ,13,14, iWe previously reported that the surface expression and channel activity of ANO1 can be regulated by ANO1-interacting proteins, such as 14-3-3\u03b3 and \u03b2-COP, in glioblastoma cells ,11. In a\u03b3 phosphorylation at serine 727 of ANO1 in cerebrovascular cells [http://gps.biocuckoo.org) and confirmed the possible CaMKII phosphorylation site(s) by site-directed mutagenesis experiments tested in these studies. Because of the different cell types and experimental conditions, it seems that they provided different serine residues of ANO1 as CaMKII-dependent phosphorylation sites. However, we can exclude the involvement of CaMKII\u03b3 in the inhibitory effect of KN-93 on ANO1 currents and the migration of U251 cells, as CaMKII\u03b3 was not expressed in U251 glioblastoma cells (Our results showed that CaMKII\u03b2 and CaMKII\u03b4 were expressed in U251 glioblastoma cells and only CaMKII\u03b2-overexpression enhanced the ANO1-mediated current in the cells . Howeverar cells  and CaMKar cells . These tma cells . UnfortuIn conclusion, the CaMKII\u03b2-mediated regulation of ANO1 surface expression plays a critical role in the oncogenic properties of glioblastoma cells. The specific regulatory mechanism of ANO1 by CaMKII\u03b2 should prove to be helpful in understanding the functional roles of ANO1 in glioblastoma cells. Eventually, this finding may advance research into efficient therapeutic targets for various ANO1-mediated cancers, including glioblastoma."}
+{"text": "This study aimed to screen osteosarcoma (OS) prognosis relevant genes for methylation dysregulation, and the functional mechanisms of FES overexpression in OS cells were investigated.The OS prognosis relevant genes with differentially methylated positions (DMPs) identified from the GSE36001 and GSE36002 datasets, and the UCSC database, were used as a training set to construct a risk model, while the GSE21257 dataset was used as validation set. The expression levels of several key genes in OS cells after 5-Aza-2\u2032-deoxycytidine treatment were detected by qPCR. The effects of FES overexpression on cell proliferation, cell cycle, migration, and invasion of MNNG/HOS were analyzed by CCK8, flow cytometry, and Transwell assays.A total of 31 candidate genes, corresponding to 36 DMPs, were identified as OS prognosis relevant genes; from these, the top 10 genes were used to construct a risk model. Following validation of the risk model, FES, LYL1, MAP4K1, RIPK3, SLC15A3, and STAT3 showed expression changes between the OS and control samples. qPCR results showed that the expression of FES was significantly downregulated in three OS cell lines and increased after 5-Aza-DC treatment. The proliferation, cell cycle progression, migration, and invasion of MNNG/HOS cells were significantly inhibited after transfection with FES overexpression plasmid, and the protein expression of FYN and \u03b2 catenin were decreased in MNNG/HOS cells by FES overexpression.The decrease in FES by hypermethylation was associated with OS prognosis, and might contribute to the proliferation, migration, and invasion of OS cells. FES, and its upstream FYN and \u03b2 catenin, might coordinately exert a tumor suppressor effect in OS cells. Osteosarcoma (OS) is a type of bone malignancy. Most diagnoses occur in children and adolescents, with a higher incidence males . It is tRecently, gene methylation, an important component of epigenetics research, has been implicated the development, progression, and prognosis of OS , 6. Oh eMethylation induced gene silencing is an epigenetic mechanism associated with the development and progression of cancers , 11. Withttp://xena.ucsc.edu/). The data from GSE36001, GSE36002, and the UCSC database were used as the training set, while the GSE21257 dataset was used as the validation set. The mRNA data and survival information of 53 OS patients were downloaded. GSE21257 data was obtained using the Illumina Human-6 v2.0 Expression BeadChip platform (GPL10295).A total of three OS datasets , downloaded from the GEO database, were utilized. A set of 19 OS cell lines and six normal samples with both methylation expression data (GSE36002) and mRNA expression data (GSE36001) were examined in this study. Methylation data were produced using the Illumina HumanMethylation27 BeadChip platform (GPL8490), while the mRNA data were generated using the Illumina Human-6 v2.0 expression BeadChip platform (GPL6102). In addition, the clinical information and mRNA-Seq data of 259 sarcomas samples were downloaded from the UCSC database (2| fold change (FC)|\u2009>\u20090.585.Before data preprocessing, the methylation signals and unmethylated signals provided in GSE36002 were extracted firstly. The \u03b2 value [methylation signal/] was calculated to indicate the methylation degree of one site. Following probe filtration, by deleting the probes with a p-value\u2009>\u20090.05 and located on sex chromosomes , a BayesBefore conducting correlation analysis, gene expression values were obtained. In brief, according to the annotations file, the mRNA probes in the GSE36001 dataset were annotated in corresponding genes. If several probes were annotated in one gene, the mean expression value of these probes was considered as the expression value of this gene. Then, the Pearson\u2019s correlation coefficient (r) between the screened DMPs from GSE36002 and their corresponding genes from GSE36001 was calculated. The genes with r\u2009<\u20090 and p-value\u2009<\u20090.05 were considered candidate genes for further analysis.The expression values of candidate genes and prognosis relevant clinical information of 259 sarcoma samples were extracted from the UCSC database, which included the data of overall survival and survival status. Then, univariate Cox regression analysis was conducted to screen candidate genes under the cutoff of p\u2009<\u20090.05.The top 10 screened candidate genes, listed by p-value, were considered as the preliminary range for risk score calculation. Based on the \u03b2 and expression values of genes, the risk score for each sample was calculated by the following equation :1\\docume\u03b2 indicates the prognostic correlation coefficient, and expr indicates the expression value of the gene. By setting median risk score as the boundary, the samples were divided into high and low risk types. The top 10 genes, based on their p-values , were added into Eq.\u00a0In this formula, The expression data and corresponding survival information from the GSE21257 dataset was used as validation data. The process was repeated, followed by the extraction of the relative data for key genes. The genes with consistent expression trends in both the training and validation sets were selected for further analysis.Following the calculation of the corresponding prognostic values of the key genes, the risk score of each sample was obtained using Eq.\u00a02, while hFOB 1.19 cells were cultured at 37\u00a0\u00b0C with 5% CO2.Three OS cell lines , and a normal human osteoblastic cell line, hFOB 1.19, were purchased from the Cell Collection of the Chinese Academy of Science . The Saos2 cells were maintained in McCoy\u2019s 5A medium supplemented with 15% fetal bovine serum (FBS) and 1% penicillin\u2013streptomycin . The MG-63 and MNNG/HOS cell lines were cultured in MEM medium supplemented with 10% FBS and 1% PS, while the hFOB 1.19 cells were cultured in D-MEM/F-12 medium supplemented with 10% FBS and 1% PS. The three OS cell lines were incubated in humidified air at 37\u00a0\u00b0C with 5% CO6 OS cells of the different lines were seeded into 6-well culture plates, respectively, followed by digestion with trypsin. After these cells were treated with 5\u00a0\u03bcM 5-Aza-DC  for 48\u00a0h, the mRNA expressions levels of several key genes were detected by RT-PCR.Approximately 3\u2009\u00d7\u200910\u2212\u0394\u0394Ct method.Total RNA from each cell line was extracted using TRIzol reagent  based on the manufacturer\u2019s instructions. The expression levels of several key genes used for risk model construction and validation in the four cells lines and three OS cell lines, before and after 5-Aza-dC treatment, were detected by qPCR analysis, respectively. The cDNA was synthesized using primeScript RT Master Mix  . PCR was then conducted using Power SYBR Green PCR Master Mix . GAPDH was applied as the internal control, and all primers sequence are presented in Table\u00a0E. coli and colonies were cultured in medium containing penbritin. To confirm transfection, PCR and western blot (WB) analysis of individual colonies, and DNA sequencing of recombinant FES overexpression plasmids were performed.Full length FES cDNA was amplified by PCR and subcloned into pCDH-CMV-MCS-EF1-copGFP-T2A-Puro lentiviral expression vectors, followed by digestion with Xba I/EcoR I. The recombinant FES overexpression constructs were then transfected into 2. Cell viability was measured after 24, 48, and 72\u00a0h post transfection using the Cell Counting Kit-8 . In brief, 10\u00a0\u03bcL CCK8 solution (5\u00a0mg/mL) was added, and the absorbance of each well at 450\u00a0nm was measured using a microplate reader , followed by dark incubation for 2\u00a0h.The MNNG/HOS cells transfected with FES overexpression plasmid, or empty vector (negative control), as well as normal MNNG/HOS cells (blank) were digested using 0.25% trypsin and centrifuged at 1500\u00a0rpm for 5\u00a0min. The cells were then seeded into 6-well culture plates and cultured in complete medium at 37\u00a0\u00b0C with 5% COg for 3\u00a0min and washed once with PBS. Afterward, the cells were fixed overnight by adding 6\u00a0mL 70% pre-cooled ethanol at 4\u00a0\u00b0C. Cells were then washed twice with PBS and resuspended in 100\u00a0\u03bcL binding buffer with adding 50\u00a0\u03bcg/mL RNase A. Following staining with 5\u00a0\u03bcL propidium iodide  at 37\u00a0\u00b0C for 30\u00a0min, the percentage of cells in the G1, S, and G2/M phases was detected by flow cytometer  and Modfit software was used to analyze the data.For cell cycle assays, at 48\u00a0h post transfection, the cells were collected through centrifugation at 3006/mL) in serum-free culture medium were seeded into the upper chamber, while complete medium with 20% FBS (500\u00a0\u00b5L) was added to lower chamber. After 48\u00a0h of incubation at 37\u00a0\u00b0C, nonmigrated cells were scraped and migrated cells were stained with Crystal Violet. Stained cells were then observed using an Olympus IX73 microscope in six randomized fields (\u00d7100). For the invasion assay, the procedures were the same, except 100\u00a0\u03bcL matrigel , diluted with basal medium without serum, was added to the upper chamber.The effects of FES overexpression on the migration and invasion capacity in MNNG/HOS cells were investigated. Cell migration and invasion were detected using Transwell chambers with polycarbonic membranes (8\u00a0mm pore filter size). For the migration assay, cells (100\u00a0\u00b5L) transfected with FES plasmid or empty vector (3\u2009\u00d7\u200910https://string-db.org/). However, the relationships between FYN, \u03b2-catenin, and FES in OS have not been evaluated. Thus, the possibility that overexpressed FES influences the protein expression of FYN and \u03b2-catenin was raised. The collected cells in each group were dissolved by RIPA lysis buffer , and the supernatants were reserved following centrifugation . The concentration of proteins in each sample was detected using the BCA Protein Assay Kit , and then the lysates were separated on 12% SDS-polyacrylamide gels. The separated proteins were transferred into PVDF membrane  and the membranes were incubated with primary antibodies at 4\u00a0\u00b0C overnight. Following, the membranes were incubated with anti-rabbit or anti-mouse horseradish peroxidase-conjugated second antibodies. The blots were viewed by the Millipore ECL system. \u03b2-actin was applied as an internal control. The primary antibodies were rabbit anti-FYN , rabbit anti-\u03b2-catenin (D10A8) XP\u00ae , mouse anti-Flag Tag , and mouse anti-\u03b2-actin .FYN and \u03b2-catenin are reported to be responsible for the development and progression of OS , 17. IntResults were presented as mean\u2009\u00b1\u2009SD. All statistical analyses were performed using Graphpad Prism 5. The comparisons of quantitative data between control and experimental groups were analyzed by the Student\u2019s t-test, and the multiple comparisons were analyzed using a two-way analysis of variance (ANOVA). A p-value\u2009<\u20090.05 was considered statistically significant.After probe filtration, a total of 26,569 probes were obtained. Then, based on the above screening threshold, a set of 501 DMPs across the OS and normal samples were screened, including 495 hyper-methylated positions and 6 hypo-methylated positions.In addition, the probes in GSE36001 were totally annotated into 19,569 genes, and above 501 DMPs were matched to 401 genes. The correlation analysis showed that only 130 DMPs were significantly negatively correlated with 114 genes, and these genes were considered candidate genes for future analysis.Based on the expression values and prognosis relevant clinical information of sarcoma samples provided in the UCSC database, a total of 31 candidate genes corresponding to 36 DMPs were identified as prognosis relevant genes though univariate Cox regression analysis.In order to screen the key genes, the top 10 prognosis relevant genes showing hypermethylation were chosen for risk score calculation. When the 10 genes were added to the risk score formula Eq.\u00a0, and theThe GSE21257 dataset was used to validate the risk model. The AUC value for high and low risk sample identification by these 10 genes was as much as 0.996  that showed consistent expression trends in different risk samples, in both the training and validation sets, were selected for further analysis. The qPCR results indicated that expression of SLC15A3, LYL1, and FES were all significantly reduced in the Saos2, MG63, and MNNG/HOS OS cell lines compared with normal hFOB 1.19 cells, while RIPK3 expression was only significantly decreased in the Saos2 and MG63 cell lines (p\u2009<\u20090.01) and no significant differences between MNNG/HOS and hFOB 1.19 were found. Compared with the hFOB 1.19 cell line, the expression of MAP4K1 was remarkably enhanced in the MG63 cell line (p\u2009<\u20090.01), but significantly reduced in the Saos2 and MNNG/HOS cell lines (p\u2009<\u20090.01). In addition, STAT3 was significantly overexpressed in the Saos2 cell line, but decreased in the MNNG/HOS cell line. No significant change in STAT3 was found between the hFOB 1.19 and MG63 cell lines , whereas after 5-Aza-DC treatment, STAT3 expression was significantly upregulated in the three OS cell lines (p\u2009<\u20090.01). In addition, the expression of LYL1 and MAP4K1 were enhanced in the MNNG/HOS cells, but reduced in MG63 cells treated with 5-Aza-DC. No significant changes in LYL1 and MAP4K1 were found in Saos2 cells. Similarly, no obvious changes in RIPK were detected in the three OS cell lines between pre- and post-5-Aza-DC treatment , is a dominant-acting oncoprotein that has tyrosine-specific protein kinase activity . It contReportedly, aberrantly activated FES is related to the proliferation, migration, and invasion of several neoplasms , 30. VoiProto-Oncogene C-Fyn (FYN) is also a Src tyrosine kinase family member, which is implicated in the regulation of the cytoskeleton, integrin signaling, and cell growth , 34. SimThe bioinformatic analysis results showed that the risk model constructed by 10 OS prognosis relevant genes could be used for OS prognosis, with high AUC value. The reduced expression of FES, induced by its hypermethylation status, may be responsible for OS progression and prognosis. In addition, the overexpression of FES could inhibit the proliferation, migration, and invasion of OS cells. FES, regulated by its upstream FYN and \u03b2 catenin, might coordinately exert a tumor suppressor effect in OS cells."}
+{"text": "KD = 0.87 nM). The confocal fluorescence microscopy revealed that peptide M49 formed a ternary complex with VEGF and its receptor, which was then internalized into human umbilical vein endothelial cells (HUVECs) via VEGF receptor-mediated endocytosis. The backbone-cyclized peptide M49K was conjugated with a drug, monomethyl auristatin E, to afford a PDC, which inhibited VEGF-induced HUVEC proliferation. HLH peptides and their PDCs have great potential as a new modality for targeted molecular therapy.As a new alternative to antibody-drug conjugates, we generated \u201cligand-targeting\u201d peptide-drug conjugates (PDCs), which utilize receptor-mediated endocytosis for targeted intracellular drug delivery. The PDC makes a complex with an extracellular ligand and then binds to the receptor on the cell surface to stimulate intracellular uptake via the endocytic pathway. A helix-loop-helix (HLH) peptide was designed as the drug carrier and randomized to give a conformationally constrained peptide library. The phage-displayed library was screened against vascular endothelial growth factor (VEGF) to yield the binding peptide M49, which exhibited strong binding affinity ( Antibodies are indisputably the most successful agents used for molecular targeted therapy , 2. HoweThe antibody-drug conjugate (ADC) technology is rapidly expanding as a cancer therapeutic approach , 9. HoweIn the ADC mechanism of action, the antibody must bind a cell surface molecule to stimulate the cell internalization by receptor-mediated endocytosis, followed by delivery to lysosomes. However, the requirement for cell internalization makes the generation of ADCs even more complex and difficult . The iso1X1X1X1X1X1X1X1X1GKLX2X2LKX2KLX2X2LKX2AC) has been displayed on the minor coat protein (pIII) of the M13 filamentous phage by modification of the pComb3 system +: 4724.69 (average isotopes), Found [M+H]+ m/z = 4724.18.The C-terminal thioesterification and NCL reaction were performed simultaneously. A linear peptide (10 mg) was dissolved in 1 mL of 200 mM Na222H355N54O57 [M+H]+: 4692.52 (average isotopes), Found [M+H]+ m/z = 4691.09.Peptide (5.0 mg) was dissolved in 1 mL of 100 mM Tris-HCl (pH 6.5) containing 200 mM VA-044, 250 mM TCEP, and 40 mM glutathione. The reaction was performed for 12 hours at room temperature. The desulfurized peptide was purified by RP-HPLC and lyophilized . Molecular mass was confirmed by MALDI-TOF-MS. Calcd for CCD spectra between 260 and 190 nm were collected with a J-820 spectropolarimeter  by using a 0.1 cm path length quartz cell. The peptides were dissolved in 20 mM phosphate buffer (pH 7.0) at 20 \u03bcM of concentration. The scan speed, response time, and bandwidth for J-820 were 50 nm/min, 2 s, and 1 nm, respectively.Each peptide was dissolved in 100 mM Tris-HCl (pH 8.5) at a concentration of 300 \u03bcM, and trypsin was also dissolved in the 100 mM Tris-HCl buffer at a concentration of 75 nM. For stability assay, 100 \u03bcL of the peptide solution and 100 \u03bcL of freshly prepared trypsin solution were mixed and incubated at 37\u00b0C. Aliquots of 20 \u03bcL were sampled at different time intervals and mixed with 60 \u03bcL of 1M HCl. The remaining peptide was analyzed by RP-HPLC.261H400N58O66S3 [M+H]+: 5501.51 (average isotopes), Found [M+H]+ m/z = 5501.08.M49K was dissolved in water at a concentration of 3 mM, and Cy5-Maleimide was dissolved in DMF at a concentration of 15 mM. M49K  and Cy5-Maleimide  were mixed and incubated for 12 hours at room temperature in the dark. Cy5-labeled M49K was purified RP-HPLC and lyophilized. Molecular mass was confirmed by MALDI-TOF-MS. Calcd for C2 incubator. For VEGFR co-localization assay, HUVECs were treated with a mixture of M49K-Cy5 (500 nM) and recombinant human VEGF-A165 (100 nM) for 1 hour at 4\u00b0C. After the wash with cold PBS, VEGFR-2 was stained by the combination of mouse anti-VEGFR2 antibody  and Alexa Fluor goat anti-mouse IgG (H+L) (Invitrogen) at 4\u00b0C. Confocal images were collected using an FV1200 laser scanning microscope (Olympus).HUVECs were expanded in EGM-2 medium (Lonza) and seeded on 35 mm glass-based dish (IWAKI) and cultured in DMEM supplemented with 10% FCS for 12 hours. After 5 times wash with PBS, each peptide sample and VEGF-Alexa488 were diluted in EBM-2 and added into HUVECs coated glass-based dish for 2 hours in a CO2 incubator. After 5 times wash with PBS, HUVECs were trypsinized for 10 min to detach from a culture dish and remove the VEGF that bound to the cell surface. The trypsinized HUVECs were neutralized using DMEM medium and analyzed by flow cytometer (BD Accuri C6).HUVECs were seeded in gelatin-coated 24-well microplate in EBM-2 supplemented with 2% FCS. After overnight incubation, HUVECs were treated with VEGF-Alexa488 (100 nM) and/or M49K-Cy5 (500 nM) for 6 hours in a COM49K  and maleimide-functionalized vcMMAE  were dissolved in 1 mL of DMF. The reaction was performed for 12 hours at room temperature. The reaction product, M49K-vcMMAE, was purified by RP-HPLC and lyophilized . Molecular mass was confirmed by MALDI-TOF-MS.165 in the presence of M49Kmut-vcMMAE (50 nM), M49K-vcMMAE (50 nM), and the mixture of M49K-vcMMAE (50 nM) and M49 (500 nM) for 24 hours at 37\u00b0C. Then, 10 \u03bcL of WST-1 reagent was added into each well. After 1 hour of incubation at 37\u00b0C, absorbance was read at 450 nm. The control condition for 100% proliferation was obtained from wells coated with VEGF stimulated HUVECs, and 0% was obtained from wells without cells. Data represent mean \u00b1 standard deviation of three independent experiments.HUVECs (5000 cells/100 \u03bcL/well) were seeded in gelatin-coated 96-well microplate in EBM-2 supplemented with 2% FCS. After overnight incubation, HUVECs were stimulated by 25 ng/mL of VEGF-AS1 File2(M49/background) was calculated using the fluorescence intensity of anti-goat IgG-Cy3 conjugate and anti-M13 antibody-Alexa647 conjugate that were indicated as green intensity and red intensity, respectively.The binding signals of M49 against all spots were presented. Normalized log(XLSX)Click here for additional data file.S1 FigThe all clones specifically bound to VEGF-A, and M49 phage clone presented the strongest binding signal to VEGF-A. The data represent the mean \u00b1 standard deviation (n = 3).(TIF)Click here for additional data file.S2 Fig+ 4682.1 (m/z), calculated [M+H]+ using monoisotopes 4682.6).The reaction was monitored by RP-HPLC. The UV profile at 280 nm is shown. The separations were performed using a linear gradient (10\u201390%) of eluent B in eluent A over 30 min . Linear M49 was converted to cyclic M49 (MALDI-TOF-MS: observed [M+H](TIF)Click here for additional data file.S3 FigCD spectra were collected at 20\u00b0C in PBS, and the concentration of peptides was 20 \u03bcM. The synthetic peptides showed typical \u03b1-helical arrangement .(TIF)Click here for additional data file.S4 FigKD values (n = 3) were 96.9 \u00b1 3.0 nM, 1960 \u00b1 20 nM, 3540 \u00b1 80 nM and 0.87 \u00b1 0.15 nM, respectively. Sensorgrams of M49 were fitted with the 1:1 Langmuir model. ka, (9.2 \u00b1 1.9) \u00d7 105 (1/Ms); kd, (7.6 \u00b1 0.1) \u00d7 10\u22124 (1/s).The binding affinity of (A) M36, (B) M41, (C) M42 and (D) M49 were determined with SPR. The sensorgrams and scatchard plots were presented. (TIF)Click here for additional data file.S5 FigVEGF-A, -C, and -D were immobilized on CM5 sensor chip at 1000 RU by using amine coupling method. (A) The sensorgrams of peptide M49 to immobilized VEGF proteins. Peptide M49 was injected at a concentration of 1 \u03bcM, and association time was 60 seconds. (B) The sensorgrams of bevacizumab at a concentration of 100 nM.(TIF)Click here for additional data file.S6 Fig(A) CD spectra of peptide M49 and its variants at 20\u00b0C (solid line) and 80\u00b0C (dash line). (B) Tryptic stability of M49 peptide and its variants. The peptides were incubated with trypsin, and the remaining peptides were analyzed by RP-HPLC. The data represent the mean \u00b1 standard deviation (n = 3). (C) The amino acid sequence of the synthetic peptides. Underlined cysteine residues are involved in disulfide bond formation. These peptides were synthesized using Fmoc-SPPS and molecular mass was confirmed by using MALDI-TOF-MS.(TIF)Click here for additional data file.S7 Fig2O/TIS, 3h, 15% yield; (b) 2 mM MPAA, 20 mM TCEP, 200 mM Na2HPO4 pH7, 12h, RT, 40% yield; (c) 200 mM VA-044, 250 mM TCEP, 40 mM Glutathione, 12h, RT, 65% yield.(a) 94:2.5:2.5:1 TFA/EDT/H(TIF)Click here for additional data file.S8 Fig165. M49K (200\u20136 nM) were injected over the VEGF-A immobilized flow cell as an analyte. Flow rate, temperature, and running buffer were 3.0 \u03bcL/min, 25\u00b0C, and HBS-EP+, respectively. The binding parameters were calculated by BiacoreT200 evaluation software with the 1:1 Langmuir model. The fitting curves were indicated as black lines. KD, 4.45 \u00b1 1.30 (nM); ka, (4.46 \u00b1 0.71) \u00d7 105 (1/Ms); kd, (2.02 \u00b1 0.75) \u00d7 10\u22123 (1/s). The data represent the mean \u00b1 standard deviation (n = 3).(A) CD spectra of M49 and desulfurized M49K at 20\u00b0C in 20 mM phosphate buffer (pH 7.0). (B) Sensorgrams of desulfurized M49K binding to recombinant human VEGF-A(TIF)Click here for additional data file.S9 Fig261H400N58O66S3 [M+H]+: 5501.51 (average isotopes), Found [M+H]+ m/z = 5501.08 (C) Sensorgram of the M49K-Cy5 binding to VEGF-A165. M49K-Cy5 was injected over the VEGF immobilized flow cell as an analyte (400\u201325 nM) at 25\u00b0C. The binding parameters were calculated by BiacoreT200 evaluation software with the 1:1 Langmuir model. The fitting curves were indicated as black lines and KD value was 5.5 nM.(A) Analytical HPLC profile of M49K-Cy5. Analytical HPLC was performed linear gradient (10\u201390%) of eluent B in eluent A over 30 min.  on a C-18 column . (B) MALDI-TOF-MS spectra of M49K-Cy5. Calculated for C(TIF)Click here for additional data file.S10 Fig(A) HUVECs were treated with M49K-Cy5 (500 nM) and VEGF-Alexa488 (100 nM) for 6 hours at 37\u00b0C. Cell nuclei were stained by Hoechst 33342 before imaging. White arrows indicate co-localized vesicles. (B) An enlarged image of (TIF)Click here for additional data file.S11 FigHUVECs were incubated for 6 hours with different solutions: VEGF-Alexa488 (100 nM), M49K-Cy5 (500 nM), and mixture of VEGF-Alexa488 (100 nM) and M49K-Cy5 (500 nM). PBS treated cells served as negative control.(TIF)Click here for additional data file.S12 Fig290H461N65O72S [M+H]+: 6042.22 (average isotopes), Found [M+H]+ m/z = 6041.81, M49Kmut-vcMMAE: Calculated for C276H452N64O71S [M+H]+: 5834.99 (average isotopes), Found [M+H]+ m/z = 5833.86 (C) Sensorgrams of the PDCs binding to VEGF-A165. M49K-vcMMAE was injected over the VEGF immobilized flow cell as an analyte (1000\u201316 nM). The binding parameters were calculated by BiacoreT200 evaluation software with the 1:1 Langmuir model. The fitting curves were indicated as black lines. KD value was 30 nM. M49Kmut-vcMMAE was injected at a concentration of 1000 nM.(A) Analytical HPLC profile of purified PDCs. Analytical HPLC was performed linear gradient (10\u201390%) of eluent B in eluent A over 30 min.  on a C-18 column . (B) MALDI-TOF-MS spectra of PDCs. M49K-vcMMAE: Calculated for C(TIF)Click here for additional data file.S13 Fig50 value of M49K-vcMMAE is 50 nM. The data represent the mean \u00b1 standard deviation (n = 3).The IC(TIF)Click here for additional data file.S1 TableAfter 4 rounds of the screening, we obtained 10 HLH peptide clones. The consensus sequences were displayed in red, and the numbers of identified clones were showed as frequency.(TIF)Click here for additional data file.S2 Table(TIFF)Click here for additional data file."}
+{"text": "Background: Metabolic syndrome (MetS) can start in children with obesity at very young ages. Non-alcoholic fatty liver disease (NAFLD) is considered to be the hepatic component of metabolic syndrome. If left untreated, the clinical course of NAFLD can be progressive and can become chronic if not detected at an early stage.Objective: We aimed to quantify the differences in liver enzymes between prepubertal children with obesity and children with normal weight to determine any associations between them and parameters related to MetS, adipokines, or markers of endothelial dysfunction and inflammation.Methods: This cross-sectional study included 54 prepuberal children with obesity (aged 6\u20139 years) and 54 children with normal weight, matched by age and sex. Liver enzymes, C-reactive protein (CRP), interleukin-6, soluble intercellular adhesion molecule-1 (sICAM-1), adipokines, and parameters related to metabolic syndrome (MetS) were all measured.Results: Alanine aminotransferase (ALT) levels, serum butyryl cholinesterase (BChE), leptin, CRP, sICAM-1, triglycerides, blood pressure, and homeostasis model assessment for insulin resistance were significantly higher in children with obesity, while Apolipoprotein A-1, HDL-cholesterol, and adiponectin were significantly lower. In the children with obesity group, ALT and BChE levels correlated with anthropometric measurements, insulin resistance, and lipid parameters, leptin, interleukin-6, CRP, and sICAM-1 while BChE levels negatively correlated with adiponectin.Conclusions: Compared to children with normal weight, prepubertal children with obesity had elevated values for liver enzymes, leptin, markers of insulin resistance, inflammation, and endothelial dysfunction, and variables associated with MetS. There was also a correlation between these disorders and liver enzyme levels. Metabolic syndrome (MetS) can start in obese children at very young ages , and a nTogether with the disorders that define metabolic syndrome, a subclinical inflammation state indicative of alterations producing endothelial dysfunction, as well as changes in adipokine levels, have been documented in prepubertal children with obesity \u20136.Non-alcoholic fatty liver disease (NAFLD) is associated with important components of MetS such as obesity , 8, someThe pathogenesis of NAFLD is multifactorial, dietary factors, insulin resistance (IR), inflammation, adipocytokines, lipotoxicity, and a genetic predisposition, are all factors that may be involved .Elevated Alanine aminotransferase (ALT) levels are associated with the incidence of MetS, diabetes mellitus, and cardiovascular disease and have been shown to be a predictive factor for non-alcoholic steatosis , 14. EleAdipose tissue plays an important role in the pathogenesis of NAFLD by contributing to the low-grade inflammation strongly related to this disease . In a suThe available data indicate that obesity-associated metabolic disorders, including NAFLD, IR, and inflammation, as well as altered levels of adipokines, can onset at very early ages. Although limited, the data on the natural history of pediatric NAFLD show that a few children rapidly progress from NAFLD to clinical events, some of which are even more severe than those diagnosed in adults , 20. ThiLimited information is available about the possible association between liver-function enzymes, obesity-related disorders, and NAFLD in prepubescent children. There is currently a lack of predictors of NAFLD among young children with obesity. The seriousness of these pathologies and the consequences of their clinical evolution without treatment serve to highlight the need for their early diagnosis and treatment. Therefore, we must try to develop diagnostic tools that can be applied in general clinical practice.Thus, in this study we aimed to analyse the possible differences between children with obesity and prepubertal children with normal weight in liver enzyme values to determine any associations between them and parameters related to MetS, adipokines, and markers of endothelial dysfunction or inflammation.We carried out a cross-sectional study in prepubertal (Tanner stage 1) children with obesity of both sexes. To reduce selection bias, both groups, cases and controls were selected from the same population, and case control ratio observed was of 1:1, matched by age and sex.Inclusion criteria were the same for the study group and control group with the exception for BMI: prepubertal (Tanner stage 1) children of both sexes and aged 6\u20139 years. The children were placed into one or the other group according to whether their BMI categorized them as with obesity (cases) or normoweight (control).Exclusion criteria for both groups: children with diabetes (fasting glucose \u2265 7.0 mmol/L), impaired fasting glucose (fasting glucose \u2265 6.1 mmol/L or < 7.0 mmol/L), primary hyperlipidaemia, hypertension, aspartate aminotransferase >40 U/L, or secondary obesity were excluded from both the study. None of the participants were receiving any regular treatments with any medications. Children with CRP levels > 10 mg/L , were excluded from both the children with obesity group and the control group. ALT levels must be interpreted in the context of the gender specific upper limits of normality in children (22 U/L for girls and 26 U/L for boys) and not upon the upper limits of normality provided by individual laboratories . ReferenAll the children in this study were Spanish. Both, children with obesity and children with normal weight were selected from the same schools, and had similar lifestyles. We informed in four schools in the area about this study in order to recruit the participants . All the parents of the children included in this study gave their written consent, and the study was authorized by the local hospital ethics committee.First, parents were invited to a briefing on this study and asked to participate in it. All parents with children between the ages of 6 and 9 were summoned. All students whose parents signed the informed consent were included in the study consecutively, by order of registration.One group included 54 children with obesity [with a body mass index (BMI) exceeding the 95th percentile in the reference tables for the Spanish population] , and theAfter a fasting for 12 h, blood samples were collected without venous occlusion from a vein in the antecubital fossa. All the samples were collected between 8:00 a.m. and 9:00 a.m., and were divided into aliquots and immediately frozen at \u221245\u00b0C until their analysis. Serum ALT, aspartate aminotransferase (AST), serum cholinesterase, leptin, adiponectin, soluble intercellular adhesion molecule-1 (sICAM), C-reactive protein (CRP), and interleukin-6 (IL-6) levels, as well as a range of MetS-related variables  were measured in all the children.Serum glucose, ALT, AST, BChE, creatine kinase (CK), total cholesterol, and triglycerides (TG) concentrations were measured using a random-access analyser  with reagents from the same manufacturer. Insulin was quantified using an Access 2-Immunoassay System . High-density lipoprotein cholesterol (HDL-c) was measured after precipitation of chylomicrons, very low-density lipoproteins, and low-density lipoprotein cholesterol (LDL-c) with phosphotungstic acid and magnesium ions. The LDL-c concentration was calculated using the Friedewald formula.IR was assessed using the homeostasis model formula for IR (HOMA-IR) based on fasting glucose and insulin concentrations: resistance = [insulin (mU/L) \u00d7 glucose (mmol/L)]/22.5. Apolipoprotein A1 (Apo A1), apolipoprotein B (Apo B), and CRP were measured by nephelometry  in a Dade Behring Analyzer II Nephelometer .Antigenic immunoassay methods were used to quantify adiponectin , sICAM-1 , leptin , and IL-6  in a microtiter plate analyser .2.Free fatty acids (FFAs) were quantified by a colorimetric enzyme assay . Blood pressure was measured with a mercury sphygmomanometer  after resting for 20 min in a supine position. The measurements were performed on three consecutive days and the mean was used in our analyses. The sphygmomanometer cuff width had to cover 2/3 of the length of the child's arm and so three cuff sizes were available for use . Weight was measured to the nearest 0.1 kg and height to the nearest 0.1 cm. BMI was calculated as the weight (kg)/height (m)F-test. The mean values of the groups were compared by applying Student's unpaired t-tests. All the results were expressed as a mean \u00b1 standard error mean (SEM) with a 95% confidence interval (95% CI). Statistical significance was set at p < 0.05. The correlation between the variables was evaluated using Pearson's correlation coefficient and regression analysis. Multivariate regression analysis was performed using the stepwise method. For each variable, potential confounders (0.05 < p < 0.2) were evaluated by analyzing the raw and adjusted regression coefficients.Statistical assessment was performed using Microstat  or GraphPad InStat software . Abnormal values (outliers) were excluded by applying Reed's method. The distribution of each variable was tested to check for deviance from the Gaussian distribution, and the equality of the variance was checked by using Snedecor's The clinical, anthropometric, and biochemical parameters related to MetS were measured in both groups . The meaThe univariate correlation analysis for MetS-related parameters for the children with obesity group is summarized in P partial = 0.0074), waist circumference , serum insulin , HOMA-IR , TG , HDL-c , and Apo A1  were independent predictive factors for ALT. For serum BChE, the age and sex-corrected BMI , waist circumference , TG , and Apo A1 , but not insulin , HOMA-IR , or HDL-c  were independent predictive factors.In the children with obesity group, age, and sex-corrected multivariate regression analysis showed that the BMI (p = 0.0007), waist circumference (p = 0.0009), insulin (p < 0.0001), HOMA-IR (p < 0.0001), and TG (p = 0.0010), and negatively correlated with HDL-c (p = 0.0121), and Apo A1 (p = 0.0126) while serum BChE positively correlated with BMI (p = 0.0007), waist circumference (p = 0.0143), insulin (p = 0.0494), HOMA-IR (p = 0.0320), and TG (p = 0.0015), and negatively with HDL-c (p = 0.0416).In the combined group , serum ALT positively correlated with BMI , CRP , sICAM-1 , and leptin  were independent predictive factors for ALT. For serum BChE, age and sex-corrected levels of IL-6 , CRP , sICAM-1 , and Apo A1  were independent predictive factors.A multivariate regression analysis was carried out for the children with obesity group. When adjusted for age and sex, levels of IL-6 (p = 0.0040), CRP (p = 0.0003), sICAM-1 (p = 0.0018), and leptin (p < 0.0001), but not with adiponectin (p = 0.5397) while BChE levels positively correlated with IL-6 (p = 0.0481), sICAM-1 , and leptin (p = 0.0047) levels, and negatively correlated with adiponectin (p = 0.0082).In the combined children with obesity and children with normal weight group, serum ALT positively correlated with levels of IL-6  is considered to be the hepatic component of metabolic syndrome . In concThe prevalence of NAFLD in children overweight or obesity ranges from 29.8 to 34.7%  and can The high prevalence of NAFLD, and its possible serious health consequences, make its early detection important because simple steatosis is reversible by lifestyle modifications, especially weight loss . Some exALT is closely related to the accumulation of fat in the liver and is considered a sensitive indicator of liver injury. It has been correlated with obesity and several components of MetS, including dyslipidaemia . Its eleAST and gamma glutamyl transferase (GGT) have not yet been independently tested as screening tools for NAFLD in children. In the context of elevated ALT, higher AST and GGT levels are associated with poorer histology . HoweverThe ALT test outcome is a continuous variable, and even fluctuation in the normal range also indicates a potential risk of metabolic disorders or cardiovascular disease in a given population . Of noteNAFLD is closely related to IR and hyperinsulinemia, which favors an increase in the levels of free fatty acids, TG, and the onset of hepatic steatosis . The groSerum BChE levels are associated with components of MetS has also been described in adolescents . In our Adipose tissue interacts with the liver and releases a series of adipokines involved in processes such as inflammation, insulin sensitivity, and NAFLD. An increase in BChE has also been observed in NAFLD. Another mechanism for these associations may be inflammation .Leptin increases IR and the production of fatty acids in hepatocytes and promotes inflammatory and fibrogenic pathways in the liver , mechaniIn this age group (6\u20139 years), we found a significant association both between ALT and BChE with markers of inflammation, endothelial dysfunction, and IR, in turn resulting in a positive correlation between liver enzymes and leptin concentration. Serum CRP levels are predictive of NAFLD and have been related to the presence and severity of liver fibrosis .The transition from simple steatosis to non-alcoholic steatohepatitis is accompanied by an additional decrease in adiponectin levels . It may The optimal age for NAFLD screening and the need for repeat screenings remain undetermined because of the lack of pediatric studies on the incidence and natural history of NAFLD in young patients. The North American Society of Pediatric Gastroenterology, Hepatology, and Nutrition (NASPGHAN) guidelines recommend the use of serum ALT levels to screen for NAFLD in children, starting from the age of 9 years .The data provided in this study describe the early onset of metabolic disorders related to obesity and NAFLD, which may suggest that screening should be initiated at ages under 10 years, particularly in children with obesity. Thus, it may be of interest to review the normal limits of ALT and to evaluate liver enzymes from prepubertal ages, as well as assessing the usefulness of other possible biochemical markers in pediatric patients. In this regard, extensive studies will be necessary in prepubertal children, including the evaluation of whether weight loss positively affects liver enzymes and its impact on other variables that accompany metabolic syndrome and NAFLD.The design of this current study did not allow us to establish if there were any sex-related differences in our population. Although this was a limitation, even after age and sex-corrected multivariate regression analysis, the main variables we studied for this age group maintained the same correlations. Only prepubertal children were studied in this current research in order to eliminate the influence of the hormonal changes that occur with puberty.Another limitation of this work was that we did not perform any imaging studies. Nonetheless, we did aim to assess whether any alterations in biochemical parameters related to obesity and NAFLD were present in children with obesity before puberty and to determine any correlations between them.Despite recent advances in the understanding of pediatric NAFLD, the evolution, and consequences of this condition are still unclear. The information from the future research would help create clinical programs for early diagnosis and intervention before significant vascular disease begins . In addiWe described an increase in liver enzyme values, markers of IR, inflammation, and endothelial dysfunction, together with the variables that define MetS and altered levels of adipocytokines in prepubescent children with obesity compared to age and sex-matched children with normal weight and there was a correlation between these alterations and liver-function enzymes.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by the Research and Ethics Commission, North Sanitary Area of Cordoba, Hospital Valle de los Pedroches, C/ Juan del Rey Calero s/n 14400, Pozoblanco, C\u00f3rdoba, Spain. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.MC is responsible for all data collection and samples of all participants at the Hospital Reina Sof\u00eda (C\u00f3rdoba). She wrote the manuscript and approved the final manuscript as presented. RV-M participated in designing the study, data analysis, interpretation, discussion, and collaborated in the writing of the manuscript. MV and RM created the sampling and supervised data collection. They participated in the interpretation and discussion of the data, reviewed, and edited the manuscript. RC is responsible and coordinator for recruiting children at the Hospital Reina Sof\u00eda (C\u00f3rdoba). LJ-R designed the study, collaborated in data analysis, interpretation, and discussion. All authors have read and approved the final manuscript and assume full responsibility for its contents.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."}
+{"text": "While single-molecule localization microscopy (SMLM) offers the invaluable prospect to visualize cellular structures below the diffraction limit of light microscopy, its potential has not yet been fully capitalized due to its inherent susceptibility to blinking artifacts. Particularly, overcounting of single molecule localizations has impeded a reliable and sensitive detection of biomolecular nanoclusters. Here we introduce a 2-Color Localization microscopy And Significance Testing Approach (2-CLASTA), providing a parameter-free statistical framework for the qualitative analysis of two-dimensional SMLM data via significance testing methods. 2-CLASTA yields p-values for the null hypothesis of random biomolecular distributions, independent of the blinking behavior of the chosen fluorescent labels. The method is parameter-free and does not require any additional measurements nor grouping of localizations. We validated the method both by computer simulations as well as experimentally, using protein concatemers as a mimicry of biomolecular clustering. As the new approach is not affected by overcounting artifacts, it is able to detect biomolecular clustering of various shapes at high sensitivity down to a level of dimers. Common to all SMLM variants is the stochastic switching of single dye molecules between a bright and a dark state. Conditions are chosen such that only a marginal portion of the molecules is in the bright state, so that single molecule signals are well separated on each frame. The final superresolution image is reconstructed from the localizations of all single molecule signals.Single Molecule Localization Microscopy (SMLM) has boosted our insights into cellular structures below the diffraction limit of light microscopy3. Application of SMLM to various plasma membrane proteins revealed the presence of nanoclusters to different degrees4. More recently concerns were raised that the stochastic activation process of the fluorophores, along with the presence of more than one dye molecule per labeled biomolecule, may lead to multiple observations of the same biomolecule in the superresolution image6. Different attempts were undertaken to approach this problem11, e.g. by merging localization bursts into one localization12, by analyzing the number of blinking events per localization cluster11, or by evaluating the spatial spread of the localization clusters7. A disadvantage of existing methods is the requirement of user-defined parameters12 or additional experiments to characterize the blinking statistics of the chosen fluorophores11. We recently developed a parameter-free method to identify global protein clustering based on a label titration approach8 , however, in case of faint bimolecular clustering the discrimination is difficult and rather subjective. Taken together, it would be helpful to provide a parameter-free quantitative assessment for the reliability of the statement, whether biomolecular nanoclusters occur in an image or not.Researchers have been particularly intrigued by the possibility to determine the spatial distribution of biomolecules in their natural environment, in most cases the intact cell. For example, models for cellular signaling are crucially affected by the spatial organization of receptor and downstream signaling molecules at the plasma membraneHere we present a method to assess biomolecular nanoclustering in two-dimensional SMLM via p-values in the framework of statistical significance tests, termed 2-Color Localization microscopy And Significance Testing Approach (2-CLASTA). The idea is to target the same biomolecule of interest with different fluorescent labels, determine the localizations in the respective color channels and calculate the nearest neighbor distances between them. The test compares the nearest neighbor distances for the recorded data with the distances for a randomized data set calculated from the measured data. As an output, the method provides a p-value for the null hypothesis that the experimental data set corresponds to an underlying biomolecular distribution, which is not significantly different from a completely random distribution as described by a spatial Poisson process. In this respect, 2-CLASTA differs from existing quantitative approaches, which typically aim at determining quantitative parameters before actually testing the mere presence of biomolecular clusters. The method is parameter-free and does not require any additional measurements nor grouping of localizations. We validated the method experimentally in cells expressing artificially clustered proteins by showing that sizes down to 2 molecules per cluster can be reliably detected.Labeling the biomolecule of interest in two different colors yields different two-color SMLM images for a random versus a clustered biomolecular distribution Fig.\u00a0. Both im13.To analyze the data, we hence opted for a strategy which is independent of prior information on label properties. The idea is to determine a randomized distribution function 14, which denotes a shift by an arbitrary distance in an arbitrary direction, assuming periodic boundary conditions  of 2-CLASTA for two clustering scenarios: i) biomolecular oligomerization , and ii) spatially extended clusters with varying load. The spatial distribution of the biomolecules and the according localization maps were generated with Monte Carlo simulations and evaluated with 2-CLASTA. We quantified the test performance via the sensitivity defined as 2 sized localization maps\u00a0 containing randomly distributed dimers, trimers, or tetramers, assigned labels of the two colors with the according blinking statistics and added localization errors. Each localization map\u00a0can be considered as a realization of a two-color superresolution experiment. The localization maps\u00a0were analyzed by 2-CLASTA, yielding a p-value for each localization map\u00a0and the sensitivity for each parameter set. We showcased the performance of the method with a simulated \u201cideal\u201d scenario, which lacks the presence of unspecific signals. We further assumed a global degree of labeling of 100%, i.e. each biomolecule was represented either by a blue or a red label, yielding localizations according to the experimentally derived blinking statistics for SNAP488 and SNAP647, respectively  allow for a sensitive detection even of dimerization, both for the \u201cideal\u201d and the \u201crealistic\u201d scenario . We next tested the influence of a reduced labeling efficiency. In general, sensitivity was found to be high even down to a labeling degree of ~20%  and an Alexa Fluor 647-conjugated antibody 15, yielding virtually identical curves  and SNAP-Surface Alexa Fluor 647 (SNAP647). dSTORM experiments were performed at alternating excitation, yielding superresolution images of the two color channels  anchor. For example, SNAP-concatemers of 4 SNAP-tag subunits would correspond to tetrameric protein oligomers. Clustering of the GPI anchor els Fig.\u00a0. For eacels Fig.\u00a0. For SNAThere is, however, a non-negligible fraction of cells which show p-values\u2009>\u20090.05, even in the case of oligomeric SNAP constructs. This effect is rather prominent for dimers and decreases with increasing degree of oligomerization. In a practical situation, however, one should note that different cells show different protein expression levels, thereby yielding a variability in the number of molecules within the region of interest. As shown in Fig.\u00a018.We present here a parameter-free method to statistically assess the question whether biomolecules are distributed randomly in two dimensions, yielding a p-value as output parameter. As for all SMLM methods, live cell applications are inherently restricted by dynamic cellular processes within the rather long recording times. The method is compatible with most fluorescence labeling techniques, as long as it is ensured that each protein molecule is connected to one color channel only: this includes fluorescent antibodies or nanobodies, tags, or low affinity binders19 would be feasible. It turns out, however, that localization maps - as they would result from a truly random biomolecular distribution - are difficult to obtain, particularly since the photophysics of organic dyes often changes with the local environment of the chromophore20. We circumvented the problem by analyzing not the images themselves, but a correlation metric between the localizations of the two color channels . In principle, also other metrics could be used for the significance test , especially for testing deviations on length scales beyond the nearest neighbors.Up to now, statistical detection of biomolecular nanoclustering demanded for obtaining the localization maps of a truly random protein distribution as a reliable standard for comparison with the experimental data. If such a distribution was available, comparative analysis such as R\u00e9nyi divergenceThe chosen analysis strategy based on correlation metrics offers the advantage that potential correlations between the two color channels can be deliberately broken, here by applying a toroidal shift to one of the two color channels. By this, the univariate spatial structure of each localization pattern is conserved, while possible correlations are removed. This provides the possibility of significance testing between the original data and the randomized control data sets as an additional advantage.22.To make the method immediately applicable, we provide a plugin for ImageJ . The experimental basis is a chromatically corrected two-color SMLM data-set analyzed by standard single molecule localization toolsIn the following, we give a brief discussion on the strengths and potential pitfalls of our approach:(i)2-CLASTA is stable against errors in two-color image registration or drift correction. As long as these errors are smaller than typical cross-correlation distances of the two color-channels, the effects on the obtained p-values are marginal.(ii)The sensitivity of 2-CLASTA is virtually unaffected by increased localization errors. Such errors do not change the physical separation of the biomolecules, but only distribute the single molecule localizations over larger areas, while preserving the correlation of the two color channels. As the method is essentially sensitive to the biomolecular separation, localization errors have no strong effect on the sensitivity.(iii)11), making it insensitive to overcounting problems. In addition, 2-CLASTA can directly be applied to single images, thereby simplifying experimental efforts compared to our previously published method of label density variation8.2-CLASTA is not impaired by blinking dye molecules, and does not require the recording of single molecule blinking statistics (as e.g. in the methods published in refs. (iv)The sensitivity of 2-CLASTA is not affected by any unknown characteristics of the clusters. No assumptions on cluster parameters  are required for the test. The test performs well even down to the detection of dimers, reflecting the smallest possible clusters.(v)2-CLASTA is stable against real life\u00a0experimental challenges: A typical experiment contains non-specific localizations, or false negatives as a consequence of insufficient degree of labeling. Also the labeling ratio of the two colors may be unbalanced. We extensively tested the influence of such issues in Monte Carlo simulations, and found that the test is very robust over a wide range of parameters.23 and hence will likely lead to a rejection of the null hypothesis. Also, care should be taken when selecting the region of interest: ideally, a central region of the cell should be chosen for analysis, avoiding cell edges and apparent vesicular structures. In principle, whenever reasonable one may further restrict the region of interest in order to specifically scrutinize subcellular structures (e.g. synapses) for the presence of local biomolecular clustering. This comes, however, at the cost of lower molecule numbers, hence reducing the sensitivity.The sample topography may influence the obtained results: Without further information, it is reasonable to assume a completely random distribution of biomolecules on a flat two-dimensional surface parallel to the focal plane as the null hypothesis of the test. Randomly distributed biomolecules on an arbitrary two-dimensional manifold, however, may lead to virtual clustering in the projection onto a two-dimensional plane. For example, invaginations of the plasma membrane or height differences near the cell borders will cause the accumulation of the detected positions of membrane proteins in the 2D projection6. It thereby addresses an interest by the community to include hypothesis testing in SMLM analysis24. By providing p-values, it makes use of the appropriate statistical parameter to test whether a specific data set is in agreement with a particular hypothesis25. Here, small p-values indicate suspicious deviations from randomness. Large p-values, in contrast, do not indicate any peculiarities in the sample; most notably, they do not prove a spatially random distribution of biomolecules. One should note that care has to be taken when interpreting the results of significance tests26. As a particular example, fishing for data sets with small p-values should be avoided.Taken together, we believe that the 2-CLASTA approach is well suited for a qualitative assessment of spatial two-dimensional biomolecular distributions, before more sophisticated methods are used to characterize the clustering quantitativelyA further application of 2-CLASTA is the analysis of co-localization of two different types of biomolecules: in this case, the two colors would be used to target the two different biomolecules. In this paper, we provide the framework to test for biomolecular association: extension towards assessment of biomolecular repulsion is straightforward and described in the Methods section.2.All chemicals and cell culture supplies were from Sigma if not noted otherwise. All reagents for molecular cloning were from New England Biolabs. HeLa cells were purchased from DSMZ (ACC 57 Lot 23) and cultured in DMEM high glucose medium (D6439) supplemented with 10% fetal bovine serum (F7524) and 1\u2009kU/ml Penicillin-Streptomycin (P4333). All cells were grown in a humidified atmosphere at 37\u2009\u00b0C and 5% COf sequence to the N-terminus of the GPI-anchor signal of the human folate receptor. To this end, we carried out PCR to amplify the SNAPN9183S sequence from pSNAPf (N9183S) with >15 nt overhangs complementary to adjacent regions of the following SNAPf copy. We then used the Gibson assembly Master Mix (E2611) following the supplier\u2019s instructions to iteratively insert multiple consecutive copies of the SNAPf sequence in frame with the GPI anchor. The resulting colonies were screened by site specific restriction digest using HindIII (R3104) to verify the number of inserted copies.For transient transfection of HeLa cells with GPI-anchored SNAP concatemers, we fused one or multiple copies of the SNAP488) and SNAP-Surface Alexa Fluor 647 (SNAP647) were from New England BioLabs. Both labels were reconstituted in water-free DMSO (276855) at 10\u2009mg/ml, aliquoted and stored at \u221220\u2009\u00b0C until used.SNAP-Surface Alexa Fluor 488 , 3% (v/v) OxyFluor , and 20% (v/v) sodium DL-lactate (L1375)488 and 1\u2009\u00b5M SNAP647 diluted in cell culture medium. After labeling, cells were extensively washed with HBSS, and fixed with 4% formaldehyde  and 0.2% glutaraldehyde (GA) for 30\u2009min at room temperature. After another series of two washing steps, we added 450\u2009\u00b5l freshly prepared STORM buffer immediately prior to imaging.Cells were transfected by reverse transfection using Turbofect  according to the supplier\u2019s instructions with Opti-MEM  as serum-free growth medium. Briefly, cells were detached from tissue culture flasks using Accutase (A6964). Subsequently, approximately 50,000 cells were mixed with Turbofect-DNA complexes and seeded on fibronectin-coated (F1141) LabTek chambers (Nunc) and incubated overnight. The following day, cells were labeled for 30\u201345\u2009min\u2009in the incubator with 50\u2009nM SNAPA Zeiss Axiovert 200 microscope equipped with a 100x Plan-Apochromat (NA\u2009=\u20091.46) objective (Zeiss) was used for imaging samples in objective-based total internal reflection (TIR) configuration. TIR illumination was achieved by shifting the excitation beam parallel to the optical axis with a mirror mounted on a motorized table. The setup was further equipped with a 640\u2009nm diode laser , a 405\u2009nm diode laser  and a 488\u2009nm diode laser . Laser lines were overlaid with an OBIS Galaxy beam combiner (Coherent). Laser intensity and timings were modulated using in-house developed LabVIEW software . To separate emission from excitation light, we used a dichroic mirror . Images were split chromatically into two emission channels using an Optosplit2 (Cairn Research) with a dichroic mirror  and additional emission filters for each color channel . All data was recorded on a back-illuminated EM-CCD camera (Andor iXon DU897-DV).2 intensity (640\u2009nm and 488\u2009nm) and 3\u20135\u2009W/cm2 (405\u2009nm); intensities were measured in epi-configuration. We selected the illumination times in ranges of 3\u2009ms\u201310\u2009ms (640\u2009nm), 3\u2009ms\u201330\u2009ms (488\u2009nm), and 6\u2009ms (405\u2009nm). Time delays between consecutive illuminations were below 6\u2009ms. The camera was read out after the 640\u2009nm and after the 488\u2009nm illumination, yielding 10 000 frames in each color channel. Thus, the total recording time for a full dataset ranged from 3 to 7\u2009minutes. Only data from those frames were included in the analysis, in which well-separated single molecule signals were observable.Typically, we recorded sequences of 20 000 frames in alternating excitation mode. Samples were illuminated repeatedly at 640\u2009nm, 405\u2009nm, and 488\u2009nm with 2\u20133\u2009kW/cm28. Single molecule localization and image reconstruction was performed using the open-source ImageJ\u00a0plugin ThunderSTORM29.We recorded calibration images of immobilized fluorescent beads after each experiment  and registered the images as described previously647 or SNAP488. To statistically quantify the blinking of\u00a0SNAP647 and SNAP488, localizations from individual label molecules were grouped and quantified in MATLAB . We determined the first frame of appearance, the total number of detections per label (N)  and the time a label is not detectable (toff).We quantified single label blinking on HeLa cells expressing GPI-anchored SNAP-monomers, using the identical illumination protocol as for 2-color dSTORM recordings. To assure sufficient separation between individual label molecules, dSTORM experiments were performed at low labeling concentrations of either SNAP(N) Fig.\u00a0, the tim14 to the positions of the red color channel 30, since for randomly distributed molecules We compared the positions of all localizations obtained in the red color channel ter Fig.\u00a0. To comp,1] Fig.\u00a0. The calcross as basis of the test statistic, cumulative distribution functions cross the To calculate a p-value from k-nearest neighbor statistics, cumulative distribution functions 15. All simulations were carried out in MATLAB  on a standard personal computer.Conceptually, simulations were performed as described previously2, reflecting approximately the size of a typical cell. For all simulations we used 75 molecules per \u00b5m2, if not mentioned otherwise.First, we simulated the underlying protein distributions for regions of 10\u2009\u00d7\u200910\u2009\u00b5m We\u00a0distributed oligomers randomly within the region of interest, and assigned n biomolecules to each n-mer position (n\u2009=\u20091 to 4). A random distribution of biomolecules is naturally reflected by the case of n\u2009=\u20091.2 . The number of biomolecules per domain was calculated from the total number of simulated molecules , the fraction of molecules inside domains , and the number of simulated domains, assuming a Poissonian distribution. Biomolecules were distributed randomly within the domains. The remaining molecules were distributed randomly in the areas outside of the domains.Circular domains with a radius of 20, 40, 60, 80, 100 or 150\u2009nm were distributed randomly onto the region of interest with adjustable number of domains per \u00b5mSecond, two different types of labels, corresponding to the two colors, were assigned randomly to the molecules according to the specified labeling ratio, assuming Binomial statistics.15 or artificial blinking statistics following a log-normal distribution. Localization errors were simulated by spreading these detections using a Gaussian profile centered on the molecule position with a width of 30\u2009nm, which corresponds to typical localization errors achieved in SMLM experiments. We assumed identical localization errors for the two color channels.Third, to simulate blinking, we assigned a number of detections to each label Fig.\u00a0. For the2 for each color channel, assuming the blinking statistics determined for SNAP488 and SNAP647. We finally considered also false positive localizations by adding a background of 1 (2) signals/\u00b5m2 for the red (blue) color channel, again with experimentally determined blinking statistics obtained in unlabeled cells.Fourth, to account for experimental errors in the \u201crealistic\u201d scenarios, we included unspecifically bound labels at a mean density of 5 labels/\u00b5mFifth, to account for stage drift in Fig.\u00a0Sixth, to account for residual chromatic aberrations in Fig.\u00a0If not mentioned otherwise, 100 simulations were performed for each experimental condition.2 region of interest, 75 molecules per \u00b5m2, a balanced labeling ratio between the two color channels, no stage drift, 30\u2009nm localization error (standard deviation), and the blinking statistics determined for SNAP488 and SNAP647 for the two color channels. For the \u201cideal\u201d scenario we simulated 100% labeling efficiency, no unspecifically bound labels and no unspecific background signals. For the \u201crealistic\u201d scenario we simulated 40% labeling efficiency, 5 unspecifically bound labels per \u00b5m2 and color channel, and 1 or 2 unspecific background signals per \u00b5m2 in the red and blue color channel, respectively.If not mentioned otherwise, we used the following set of parameters: 10\u2009\u00d7\u200910\u2009\u00b5mSupplementary Information.Supplementary Information2."}
+{"text": "The management of cancer surgeries is under unprecedented challenges during the COVID-19 pandemic, and the breast cancer patients may face a time-delay in the treatment. This retrospective study aimed to present the pattern of time-to-surgery (TTS) and analyze the features of breast cancer patients under the different stages of the COVID-19 pandemic.Patients who received surgeries for breast cancers at West China Hospital between February 15, 2020 and April 30, 2020 (the outbreak and post-peak stages), and between March 10, 2021 and May 25, 2021  were included. TTS was calculated as the time interval between the pathological diagnosis and surgical treatment of breast cancer patients. And the pandemic was divided into three stages based on the time when the patients were pathologically diagnosed and the severity of pandemic at that time point. TTS, demographic and clinicopathological features were collected from medical records.p<0.001) and regular screening (p<0.001), as well as age (p=0.013) and menstrual status (p=0.004). As for clinicopathological features, axillary involvement (p=0.019) was a factor that differed among three stages. The overall TTS was 23.56 \u00b1 21.39 days. TTS for patients who were diagnosed during the outbreak of COVID-19 were longer than those diagnosed during pandemic post-peak and normalization stage (p<0.001). Pandemic stage (p<0.001) and excision biopsy before surgery  were markedly correlated with the TTS of patients.A total of 367 patients were included. As for demographic features, it demonstrated statistically significant differences in insurance type (TTS of breast cancer patients significantly varied in different stages of the COVID-19 pandemic. And breast cancer patients\u2019 daily lives and disease treatments were affected by the pandemic in many aspects, such as health insurance access, physical screening and change of therapeutic schedules. As the time-delay may cause negative influences on patients\u2019 disease, we should minimize the occurrence of such time-delay. It is vital to come up with comprehensive measures to deal with unexpected situations in case the pandemic occurs. The COVID-19 pandemic caused by the novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a public health emergency of global concern, and is unprecedentedly threatening the contemporary healthcare systems (Time-to-surgery (TTS) refers to the time interval between diagnosis and therapeutic surgery. Longer TTS is associated with poorer outcomes in patient with breast cancers , 4 and oThe management of breast cancer demands special considerations during the pandemic. Although the existing studies conducted on TTS changes among patients with breast cancer during COVID-19 are mainly cross-sectional, the available evidence confirmed a time-delay in cancer treatment. High mortality risk diseases, such as cancers should not be neglected during the pandemic \u201313 and pThe study was approved by institutional review board . Informed consent was waived due to the retrospective nature of this study.Between February 15, 2020 and April 30, 2020, and between March 10, 2021 and May 25, 2021 , patients with breast cancer who received surgeries at West China Hospital of Sichuan University were retrospectively identified from hospital database. The inclusion criteria were patients with breast cancers who received surgeries. The exclusion criteria were as follows: a. male patients; b. Paget\u2019s disease; c. patients who received neoadjuvant chemotherapy; d. patients during pregnancy or lactation; e. recurrent or metastatic diseases; f. patients who have been diagnosed with other malignancies; g. patients with known major disabling medical or mental disorders.The following information of each included patient was collected: demographic, clinical, pathological features and treatment strategies.Conventionally, TTS was calculated as the time interval between the pathological diagnosis and surgical treatment of breast cancer patients. According to TTS, patients were categorized into two groups: TTS < 40 days and TTS \u2265 40 days , 14.Based on the time when the patients were pathologically diagnosed and the severity of pandemic at that time point, we categorized the patients into three groups: outbreak (diagnosed before 1.24 in 2020), post-peak (diagnosed after 1.24 in 2020) and normalization (diagnosed in 2021). As the time point of the suspension of operations, January 24, 2020 was regarded as the cut-off point for pandemic in 2020, which divided the pandemic into the outbreak and the post-peak stage. To be specific, the post-peak stage refers to a period when there was initial progress in containing the virus.t-test and one-way analysis of variance (ANOVA) for continuous data; Pearson\u2019s Chi-square test or Fisher\u2019s exact test for categorical data to compare demographic, clinical and pathological features in different periods of the pandemic; binary logistic regression for univariate and multivariate analysis of TTS. A two-sided p-value less than 0.05 was regarded as statistically significant. All statistical analyses were performed using SPSS 21.0 .We used independent p<0.001). With the progression and control of the pandemic, TTS showed a pattern of first increasing, then deceasing, before finally stabilizing. The mean TTS was 58.55 \u00b1 24.87 days during the outbreak stage of the pandemic, 16.55 \u00b1 11.10 days in post-peak stage, and 22.38 \u00b1 20.43 days in the normalization stage. The differences and patterns of TTS are demonstrated in The overall mean TTS was 23.56 \u00b1 21.39 days. The TTS was distinctly different at different pandemic stages (p<0.001) and regular screening (p<0.001). During the normalization stage, more patients paid medical bills via governmental insurance and received periodic screening than patients in other stages. Patients seen in the normalization stage were generally older (p=0.013), and more likely to be menopausal (p=0.004). Detailed demographic characteristics of the patients are presented in In total, 367 consecutive patients met the inclusion criteria and were included for statistical analysis. None of the patient was infected with SARS-CoV-2. The mean age at diagnosis was 50.74 \u00b1 10.63 years. The demographic features, such as setting, educational level, and marital status were not significantly different among the pandemic stages. Interestingly, statistically significant differences were found in the insurance type (p=0.019). Other clinicopathologic features, such as method of detection, type of surgery, AJCC stage and molecular subtype, were similar among 3 stages. Detailed clinical-pathological characteristics of the patients are presented in A significantly higher proportion of patients exhibited axillary involvement in post-peak stage than outbreak stage. On the other hand, following initial control of the virus the number of patients with axillary involvement decreased markedly during the normalization stage (p<0.001), excision biopsy before surgery , and menstrual status  were related to the TTS (p<0.001), and excision biopsy before surgery  were markedly correlated with TTS. The correlation pattern between these two factors and TTS was consistent with that in the univariate analysis.Results from the univariate analysis showed that pandemic stages . Starting from mid-February, surgeries were gradually resumed on the basis of limited admissions. By April 2020, the daily running of the hospital (diagnosis and treatment) had generally returned to normal.The TTS of patients with breast cancer was the longest during the COVID-19 outbreak stage and the shortest at the post-peak stage. In contrast, the TTS during the normalization stage in 2021 was moderate. In the beginning of the pandemic, transportation between cities and provinces were temporarily cut off to stop the virus from spreading, which led to expected diagnostic and treatment delays. Moreover, little was known about the virus at the start of the pandemic, and the fear of COVID-19 led many to voluntarily and completely avoid social contact, which may be the main reason for the increased TTS.Surprisingly, the TTS dropped shortly after the outbreak stage of the pandemic, to a duration shorter than that in the normalization stage. This may be due to patients choosing to attend local hospitals instead of waiting to be admitted in large hospitals. Further, due to the limited access to surgeries at the time, selected patients with operable breast cancers received neoadjuvant chemotherapy instead of surgeries. Consequently, the number of patients waiting for treatment in the hospital reduced, and the patients might be seen and treated more quickly during post-peak stage. The TTS of the normalization stage in 2021 is likely to be maintained for a long time into the future. Many previous studies have reported on the delay associated with cancer surgeries during the COVID-19 pandemic. The time-delay ranged from 1\u22122 weeks to several months. The risk of patient exposure, turnover times, the patient\u2019s fear, and economic instability could all influence the TTS , 9, 15. Age was a key patient characteristic that was significantly different among the different stages of the pandemic. Patients in the normalization stage were on average, older. Concerning the aging process, older patients with breast cancer tend to have more comorbidities and worse physical conditions than the younger patients . It is pMost of the potential influencing factors of TTS turned out to be irrelevant variables. Meanwhile, different stages of the pandemic accounted for most of the differences in the TTS. During the different stages of the pandemic, not only did the hospital and surgeries run differently, but the patients also experienced different events and possessed different mindsets. In the initial stage of the pandemic, the suspension of hospital operations, diminished healthcare resources, and patient\u2019s resistance to social contact all contributed to the longer TTS. Garcia, et\u00a0al. also found that delays in oncologic surgery negatively affected many tumor types . While tSome experts have suggested that it is appropriate to delay surgery during COVID-19. The European Society for Medical Oncology (ESMO) recommended that the initial operation of low-risk early breast cancer could be postponed for up to 12 weeks . Tseng, During the COVID-19 pandemic outbreak, a lack of adequate advanced preparation had led to time-delays in cancer treatment. Breast cancer centers should ensure thorough protective measures, adequate social distancing, and quali-quantitative standard of breast cancer care to be well-prepared for potential and anticipated challenges. Our study provided a perspective of how the COVID-19 pandemic affected patients with breast cancer by prolonging the TTS, which in turn inspired us to develop comprehensive methods for challenges such as a pandemic.There are several limitations to this study. Firstly, the TTS can be largely affected by the severity of local epidemics and local government policies. Thus, our findings must be interpreted with the local conditions in mind. Secondly, the retrospective design of this study may inevitably bias patient selection. Thirdly, this is a single-center study, and the sample size was relatively small.In conclusion, the current study presented the pattern of TTS for patients with breast cancers during the COVID-19 pandemic and shared the experience of a single center. Future studies with larger sample populations are warranted to validate our results.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.JC, RC, and JY contributed to conception and design of the study. RC and JY performed the statistical analysis. RC and JY wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted versionThis study is supported by the funding from the National Natural Science Foundation of China (32071284) and the Fundamental Research Funds for the Central Universities (2021SCU12021).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by increased activation of fibroblasts/myofibroblasts. Previous reports have shown that IPF fibroblasts are resistant to apoptosis, but the mechanisms remain unclear. Since inhibition of the mitochondrial permeability transition pore (mPTP) has been implicated in the resistance to apoptosis, in this study, we analyzed the role of mitochondrial function and the mPTP on the apoptosis resistance of IPF fibroblasts under basal conditions and after mitomycin C-induced apoptosis. We measured the release of cytochrome c, mPTP opening, mitochondrial calcium release, oxygen consumption, mitochondrial membrane potential, ADP/ATP ratio, ATP concentration, and mitochondrial morphology. We found that IPF fibroblasts were resistant to mitomycin C-induced apoptosis and that calcium, a well-established activator of mPTP, is decreased as well as the release of pro-apoptotic proteins such as cytochrome c. Likewise, IPF fibroblasts showed decreased mitochondrial function, while mPTP was less sensitive to ionomycin-induced opening. Although IPF fibroblasts did not present changes in the mitochondrial membrane potential, we found a fragmented mitochondrial network with scarce, thinned, and disordered mitochondria with reduced ATP levels. Our findings demonstrate that IPF fibroblasts are resistant to mitomycin C-induced apoptosis and that altered mPTP opening contributes to this resistance. In addition, IPF fibroblasts show mitochondrial dysfunction evidenced by a decrease in respiratory parameters. Idiopathic pulmonary fibrosis (IPF) is an aging-associated, progressive, and irreversible disease of unknown etiology. Currently, therapeutic options remain limited, and there is no efficient treatment to improve expectancy and quality of life ,2. FurthFibroblasts and myofibroblasts are the critical effector cells in tissue fibrosis due to their participation in elaborating the extracellular matrix and contraction mechanisms and remodeling of damaged tissue . During Mitochondria are central organelles that participate in vital metabolic processes, synthesize most ATP, and regulate several signaling cascades, including apoptosis . The tra1F0\u2013ATP synthase as the inner membrane pore-forming unit of the mPTP i by activating the mPTP in control and IPF fibroblasts. Our results indicate that FCCP elicited in normal fibroblasts a transient moderate increase in [Ca2+]i to 115.76 \u00b1 9.53 and 108.09 \u00b1 7.01 nM, respectively . To corroborate that the increase in [Ca2+]i is due to the activation of the mPTP with FCCP, we blocked pharmacologically the mPTP with cyclosporin A, inhibiting the response , which are very susceptible to cell death , an inhibitor of the opening of mPTP, and then were stimulated with mitomycin C (25 \u03bcg/mL) for 24 h. Our results showed an increase in the number of positive apoptotic cells when we used mitomycin C. Interestingly, the administration of BKA decreased the percentage of apoptotic cells in control fibroblasts (** ll death A. On thell death B.2/s/106 cells/mL) was determined in 2 \u00d7 106 non-permeabilized fibroblasts. p \u02c2 0.01), which was suppressed by oligomycin, a specific inhibitor of mitochondrial ATP synthase and oxidative phosphorylation. The proton leak was slightly increased in the IPF fibroblasts, but the difference against control fibroblasts was not significant (p = 0.06). The maximal respiration of fibroblasts was determined after the uncoupling of mitochondria with CCCP. The results showed that IPF fibroblasts had a decreased maximal respiration compared with the control group (** p \u02c2 0.01). Finally, respiratory control (RC) was calculated by the difference between basal respiration and proton leak. The results showed that IPF fibroblasts exhibited lower RC than normal fibroblasts (*** p \u02c2 0.01). Together, our findings indicate impaired respiration in IPF fibroblasts. We evaluated the oxygen consumption in permeabilized fibroblasts to determine whether the modification of respiration in IPF fibroblasts was associated with an alteration in complex I and II of the electron transport chain. We determined the electron transport chain activity using specific substrates to complex I  and complex II (succinate). A representative trace is shown in p \u02c2 0.01) or complex II (* p \u02c2 0.05)  D.As we detected alterations in oxygen consumption, we turned our attention toward the mitochondrial membrane potential: the electrochemical force that modulates the kinetics of proton re-entry to the matrix through ATP synthase. Using confocal microscopy, we measured the \u0394\u03a8m of control and IPF fibroblast stained with JC-1 (4 \u03bcM). JC-1 is a cationic fluorescent dye that exhibits potential-dependent accumulation in mitochondria A. We didDiverse studies have proposed the participation of ATP synthase in the formation of mPTP. For this reason, we evaluated the levels of expression of ATP synthase by Western blot in control and IPF fibroblasts, and no differences were found C. Furthep \u02c2 0.01). In addition, decreased mitochondrial respiration in IPF fibroblasts was correlated with a decrease in ATP production . As shown in the fluorescence micrograph with MitoTracker, we observed a mitochondrial network fragmented and accumulation of mitochondria in IPF compared to control fibroblasts. Moreover, fibroblasts from control lungs showed branched, elongated, and lengthwise organized mitochondria with a typical appearance; the cristae were electrodense and compact. In contrast, in IPF fibroblasts, the mitochondria were scarce, thinned, and disordered; the cristae showed a lesser electron density than control fibroblasts A. As sho 250 nm2 B. We als 250 nm2 C.We also determined by confocal microscopy, through a Z-stack analysis, the mean fluorescence intensity of the MitoTracker in whole cells in control and IPF fibroblasts. In addition, sections were made throughout the entire cell to indirectly assess whether there were changes in mitochondrial mass. Our results show no significant change in the fluorescence of the mitochondrial marker MitoTracker between control and IPF fibroblasts, suggesting that there are no significant differences in the number of mitochondria . In addiA growing body of evidence demonstrates that IPF fibroblasts are resistant to apoptosis ,8,9,10. In this work, we demonstrated that IPF fibroblasts presented a marked decrease in basal cytochrome c levels and its release after stimulation with mitomycin C. Cytochrome c is a crucial signaling molecule during apoptosis, but it also plays an essential role in oxidative phosphorylation by transferring electrons from complex III to complex IV of the electron transport chain . A substInterestingly, embryonic fibroblasts derived from mice deficient in the somatic isoform of cytochrome c are resistant to apoptosis by agents known to trigger the intrinsic apoptotic pathway. This effect was associated with respiratory chain defects . The mit2+ uptake, respiratory inhibition, generation of ROS, mitochondrial swelling, and potentially the rupture of the outer mitochondrial membrane leading to release of mitochondrial apoptogenic proteins such as cytochrome c, Smac/DIABLO, endonuclease G, and AIF. Therefore, it is not surprising that studies of the mPTP attract substantial attention. However, despite significant effort, the exact molecular composition of the mPTP is still a matter of debate.Induction of the mPTP leads to mitochondrial depolarization, inhibition of ATP synthesis, Ca2+, oxidative stress, and Pi and can be inhibited by cyclosporin A, adenine nucleotides, Mg2+, acidic pH, and reducing agents in the cells . When the ANT is stabilized in the cytosolic conformation by carboxyattrayloside, it provides a structural basis for mPTP formation by increasing sensibility to [Ca2+]. While on the other hand, bongkrekic acid, an inhibitor of ANT that stabilizes the matrix conformation, inhibits mPTP opening by decreasing the sensitivity to [Ca2+]  . This efOther factors that influence the opening of the mPTP may be related to cytochrome c interaction with lipids of the inner mitochondrial membrane or the capacity to accumulate calcium in the mitochondria, as calcium is a well-established mPTP activator. The reduction of mitochondrial calcium release induces mPTP inhibition.2+ accumulation in mitochondria, stabilizes mitochondrial membrane potential, and defers Ca2+ dysregulation i changes induced by trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP) in control and IPF fibroblasts, cells were plated in round coverslips coated with poly-L-Lysine and cultured for 2 days. Then, cells were loaded with Fura 2-AM (2.5 \u03bcM) in a low concentration of Ca2+ (0.1 mM) and at room temperature. Then, fibroblasts were incubated for 1 h at 37 \u00b0C under a 5% CO2 atmosphere. Afterward, cells were transferred to a heated perfusion chamber (37 \u00b0C) mounted on an inverted Nikon Diaphot 200 microscope . Cells were recorded under continuous perfusion and carbogen bubbling (to maintain pH at 7\u20134) at a rate of 2\u20132.5 mL/min with Krebs\u2013Ringer buffer  at 37 \u00b0C. After the recording of basal fluorescence, cells were exposed to FCCP 1 \u03bcM. Next, fibroblasts loaded with Fura 2-AM were alternately submitted to Xe lamp at 340 nm and 380 nm excitation light, and the emission fluorescence was measured at 510 nm using a microphotometer (model D-104), from Photon Technology International . Fluorescence was measured at intervals of 0.5 s for 10 min, and the intracellular Ca2+ concentration ([Ca2+]i) was calculated according to the Grynkiewicz i i . These pe 386 nM . To evalThe oxygen consumption experiments in intact cells were performed as previously described using a high-resolution respirometry O2 k meter  . Briefly5 fibroblasts were added, and the system was equilibrated again for 5 min. The reaction was carried out in respiration medium  pH 7.1, adjusted with 5 N KOH. Next, the fibroblasts were permeabilized with digitonin (20 \u03bcg/mL) and incubated for 5 min at 37 \u00b0C. After the addition of digitonin, the respiration rate should markedly decline for 3\u20135 min. Then, glutamate/malate (10 mM/5 mM) was used as a substrate for complex I activity, ADP was added to induce ATP production, and the respiration was inhibited by 0.5 \u03bcM rotenone, which is a specific inhibitor of complex I. Afterward, succinate (10 mM) was used as a substrate to induce complex II supported respiration, ADP was added to induce ATP production, and respiration was inhibited with oligomycin (5 \u03bcM) (state 4). Finally, 2 mM ADP was added for maximal (state 3) mitochondrial respiration. The respiratory control ratio (RCR) was calculated by dividing state 3/state 4 respiratory values.The oxygen consumption in permeabilized fibroblasts was performed as was previously described by Kuznetsov et al. . ControlMitochondrial \u0394\u03c8 was measured using JC-1, which is a fluorescent dye that exhibits potential-dependent accumulation in mitochondria. Polarized mitochondria are marked by punctate orange-red fluorescent staining. On depolarization, the orange-red punctate staining is replaced by diffuse green monomer fluorescence. Thus, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio. Briefly, IPF and control fibroblasts were stained with JC-1 in Ham F-12 culture medium for 30 min at 37 \u00b0C in the dark. Then, cells were washed twice with PBS and stimulated with the uncoupling agent FCCP (100 \u03bcM) or oligomycin (5 \u03bcM). Fibroblasts were observed with a confocal microscope using the 20x objective, and both green (520 nm) and red (572 nm) fluorescence were measured to detect the emission shift. This shift was calculated as the red/green fluorescence intensity ratio. Images were analyzed using ImageJ software.ATP production was determined quantitatively in IPF and control fibroblasts using the ATP Determination Kit, which uses a bioluminescent assay. For this assay, 10,000/well cells were plated on a white 96-well microplate with a clear bottom, and the assay was performed following the manufacturer\u2019s instructions. The bioluminescence was recorded using a multimodal microplate reader  in the luminometer mode.2 in a white 96-well microplate with a clear bottom. Following overnight incubation, the ADP/ATP ratio was determined following the manufacturer\u2019s guidelines. Next, the bioluminescence was measured using a multimodal microplate reader .The ADP/ATP ratio in IPF and control fibroblasts was measured using the ADP/ATP ratio assay kit. Briefly, IPF and control fibroblasts were plated at a density of 10,000 cells/cmTEM was done with the support of the Center for Advanced Microscopy (Northwestern University). In brief, lung fibroblasts were cultured on Thermanox coverslips placed in a 24-well plate. After 48 h of incubation, samples were fixed in 0.1 M sodium cacodylate (pH 7.2) containing 2% paraformaldehyde and 2.5% glutaraldehyde and post-fixed with 2% osmium tetroxide and stained with 3% uranyl acetate. Samples were dehydrated in ascending ethanol grades, transitioned with a 1:1 mixture of ethanol and resin, and embedded in the resin mixture of the EMbed-812 kit. Using a Leica Ultracut UC6 ultramicrotome, ultra-thin sections (70 nm) were collected on 200 mesh copper grids and post-stained with 3% uranyl acetate and Reynolds lead citrate. Ultra-thin sections were used to obtain images from each sample with the Tecnai G2 Spirit transmission electron microscope  at 120 kV. The length of mitochondria was used to determine mitochondrial elongation mean per image; at least 70 mitochondria per condition were measured. Images were analyzed using the ImageJ software.t-test. p-values of less than 0.05 were considered to be statistically significant. Statistical analysis was performed using the GraphPad Prism 5.01 software .All the data were expressed as the mean \u00b1 standard error mean (SEM). Two-way or one-way analysis of variance (ANOVA) followed by Bonferroni test assessed the significance of the differences. To analyze the significant differences between the control and IPF group, we used an unpaired"}
+{"text": "LKB1 gene and induced LKB1 expression. Our study highlights a novel mechanism by which testosterone modulates NAFLD development by inducing the mRNA expression of LKB1.The incidence of non-alcoholic fatty liver disease (NAFLD) increases in males aged >45 years, which indicates that androgens are associated with the development and/or progression of NAFLD, although excess dietary intake is the primary causative factor. However, it is uncertain how androgens are involved in the metabolic process of NAFLD, which is associated with the state of steatosis in hepatocytes. To investigate whether androgen receptor (AR) signaling influences NAFLD development, the state of steatosis was monitored in mouse livers and hepatocytes with or without androgens. As a result, hepatic lipid droplets, expression of AR, and phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) increased in the presence of testosterone. Concurrently, the expression of LKB1, an upstream regulator of AMPK, was increased by testosterone treatment. We observed that the fluctuation of AMPK-ACC signaling, which plays an important role in lipogenesis, depends on the presence of testosterone and AR. Additionally, we demonstrated that testosterone bound AR was recruited to the promoter of the  Non-alcoholic fatty liver disease (NAFLD) refers to the state of steatosis, which is characterized by the excessive accumulation of triglycerides (TGs) in hepatocytes, regardless of alcohol assumption ,2. NAFLDAdditionally, sex differences have been mentioned as another risk factor. Statistical studies have consistently revealed that the incidence of NAFLD is higher in males than in females . In maleLKB1 is decreased in adipocytes, depending on the concentration of the testosterone, which stimulates AMPK phosphorylation [LKB1 increased in the presence of testosterone in hepatocytes. Concurrently, a decrease in AMPK-ACC signaling and lipid accumulation was observed. This evidence suggests that testosterone, one of the androgens, suppresses NAFLD development as it plays an important role in the induction of transcription and/or translation of LKB1.Next, we focused on liver kinase B1 (LKB1), which is an upstream regulator of AMPK . LKB1, erylation . Interesp < 0.05) and the HD-ODXT group  (p < 0.05) like the increased hepatic TG accumulation  and were injected with 2.5 mg/mL testosterone (T) every 3 days. In addition, we divided the mice into the normal diet group (ND) and the high-fat diet group (HD) to confirm whether androgens participate in lipid metabolism A. Hemato < 0.05) B,C. In amulation D.p < 0.05) and HD-ODXT  mice (p < 0.05) and HD-Na\u00efve vs. HD-ODX  (p < 0.05) and HD-Na\u00efve vs. HD-ODX . Furthermore, the IL-\u03b2 level of HD-ODXT decreased compared to HD-ODX  , a general marker of hepatocellular injury , were si05) mice A. The mR < 0.05) B. Simila < 0.05) B. p < 0.05) and HD-ODXT mice  (p < 0.05) and HD-ODXT  groups, similar to the TGF-\u03b2 mRNA levels  C. When wA levels D. These p < 0.05)  (p < 0.05). However, the significance of pACC/ACC was not clear (p < 0.05) (p < 0.05)     A. The pAot clear A. These  < 0.05) B. Additi < 0.05) B. To fur < 0.05) C. In the < 0.05) C. This ip < 0.05) (p < 0.05) (p < 0.05)  A. Expect < 0.05) A.Concurr < 0.05) A. To ver < 0.05) B. This sp < 0.05) and HD-ODXT mice  (p < 0.05) and HD-ODXT mice   A. Simila < 0.05) A. AR; 1.37 fold, p < 0.05)     was added to the SNU-423 cells, unexpectedly, the mRNA levels of AR were increased, rather than suppressed, compared to the testosterone alone-treated cells vs. the vehicle-treated cells in the presence of bicalutamide  and in the testosterone alone-treated cells vs. the testosterone-treated cells in the presence of bicalutamide  (p < 0.05) and in the testosterone alone-treated cells vs. the testosterone-treated cells in the presence of bicalutamide    and in the testosterone alone-treated cells vs. the testosterone-treated cells in the presence of bicalutamide        B,C. To f < 0.05) B. Nevert < 0.05) C. Along  < 0.05) B,C. Thenobserved D,E. Mean < 0.05) D,E. ThisLKB1 gene by performing a chromatin immunoprecipitation (ChIP) assay  assay A. We fou\u2019 sample B. Meanwheraction B. These The incidence of NAFLD has increased in modern society, with an increasing population of individuals with obesity . This suWhen mice were fed a fat-rich diet, body weight and hepatic TG accumulation concurrently increased than the mice fed with a normal diet. The increase of hepatic TG accumulation was prominent in orchiectomized mice, compared to na\u00efve mice and the mice injected with testosterone after castration. Meanwhile, unlike the increased TG accumulation, body weight was lowest in the orchiectomized mice, among the mice fed with a fat-rich diet. It is considered that testosterone suppresses hepatic TG accumulation and increases the body weight due to an increase in fat-free mass . Along wAR overexpression in vitro assay. Concurrently, the mRNA levels of AR increased in vivo when androgens levels were sufficient. In addition, the mRNA levels of AR increased in testosterone-treated cells. The AR mRNA levels surged more sharply when cells were treated with bicalutamide, an AR antagonist. This increase is considered a compensatory response to the steep suppression of AR activation [First, we observed the fluctuation of hepatic DNL, as a recent study showed that hepatic TG accumulation is induced via DNL in patients with NAFLD . AMPK-ACtivation , as the AMPK gene, we performed a ChIP assay using the SNU-423 cells treated with testosterone. However, the results of the ChIP assay did not show the recruitment of the AR complex to the promoter of the AMPK gene. This result suggests that testosterone does not directly modulate AMPK expression. Despite the evidence, we could not exclude the genomic response of testosterone since the increased AMPK phosphorylation paralleled activation of the AR. To assess whether the complex bound with testosterone and AR (AR complex) is directly recruited to the promoter of the LKB1 mRNA expression increased when androgens and/or testosterone were present at sufficient levels both in vivo and in vitro, and in turn, the protein expression increased in vitro. Along with the increased LKB1 expression, the phosphorylation of AMPK and ACC was also raised. This implies that LKB1 mediates the signaling between androgens and AMPK phosphorylation in hepatocytes. Likewise, we speculated whether androgens drive LKB1 expression in a genomic manner and performed a ChIP assay to assess the expression of the LKB1 gene. The results showed that the AR complex was recruited to the promoter of the LKB1 gene. As in previous ChIP studies on AR, the AR element sequence motif at the AR binding site is presently presumed to be \u201cACATTTGT\u201d in part of the LKB1 gene promoter [A recent study revealed that LKB1, an upstream regulator of AMPK, interacts with sex steroids in a genomic manner in adipocytes . Therefopromoter .LKB1 transcription through direct recruitment to the promoter of the LKB1 gene. In turn, increased LKB1 expression modulates the phosphorylation of AMPK-ACC signaling, leading to the suppression of hepatic TG synthesis and accumulation. Therefore, these findings are meaningful because androgens deficiency might be another risk factor of NAFLD in males, and the control of hormone homeostasis prevents NAFLD from progressing to steatosis-induced NASH, cirrhosis, and hepatocellular carcinoma.In summary, our study suggests that androgens, particularly testosterone, can prevent hepatic TG accumulation by suppressing hepatic DNL. Androgens bound to AR modulate Antibodies used in the present study were shown in \u00ae Rodent Diet 5001 contains as a percentage of total calories: 28% protein, 60% carbohydrate, and 12% fat) and water in the manner of ad-libitum up to 8 weeks aged. To remove endogenous androgens, mice were orchiectomized bilaterally at age of 8 weeks. After the operation, we provided a high-fat diet  and injected testosterone dissolved in corn oil, 2.5 mg/mL every 3 days during 4 weeks, from recovery period for 2 weeks. After6 weeks from the operation, the mice were sacrificed. The number of mice used for the experiment was 4, 4, 4, 5, and 4 for each group: Normal diet-Navie (ND-Naive), Normal diet-Orchiectomized (ND-ODX), High fat diet-Navie (HD-Naive), High fat diet-Orchiectomized (HD-ODX), and High fat diet-ODX-treated testosterone (HD-ODXT).C57BL/6N WT male mice were accommodated and experimented in the pathogen-free facility at Chungnam National University in line with the Chungnam National University Animal Care Committee (CNU-00606). We provided them with a standard chow diet  by DRI-CHEM4000 . A portion of mice livers was fixed in 10% buffered formaldehyde solution and embedded in paraffin. The paraffin samples were sectioned to 4 \u00b5m and attached on silane coated slide. Following de-waxed and re-hydrated, the samples were stained with hematoxylin and eosin (H&E) or Masson\u2019s trichrome . The stained samples were examined using VM600 Digital Slide Scanning System . Frozen liver tissues were sectioned by 7 \u00b5m after being embedded with OCT compound and attached to silane coated slide. Following drying for 10 min in RT, slides were fixed with formalin for 20 min. Slides were washed with tap water for 10 min and subsequently rinsed with 60% isopropanol. Staining was processed with Oil-Red-O solution (3 g/L) for 15 min. After washing with 60% isopropanol, slides were stained with hematoxylin for 30 s and washed with distilled water. The stained samples were examined using a light microscope after being mounted in an aqueous mounting medium.To reveal the relationship between a level of AR expression and LKB1, two types of cell lines were used in this study. One is the SNU-423 cell line acquired from the Korean Cell Line Bank  and the other is Hep3B . Unlike the normal property of liver cells including Hep3B, SNU-423 possesses a significant level of AR expression. 2 atmosphere. SNU-423 was cultured by RPMI 1640  with 5% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S) and Hep3B by Dulbecco\u2019s modified Eagle\u2019s medium(DMEM)-High glucose  with 5% FBS and 1% P/S. Additionally, a starvation solution which is DMEM/F-12  with 2% charcoal dextran fetal bovine serum (CD-FBS) and 1% P/S, was used to deplete endogenous steroid hormones.Both cell lines were grown at 37 \u00b0C in a 5% COFatty acids , dissolved in absolute ethanol , were treated and incubated for 24 h, to provide a similar condition to the mice fed high-fat diet. Testosterone  and bicalutamide , dissolved in dimethyl sulfoxide (DMSO) , were supplemented with concentration 10 nM/L and 5 uM/L [AR expression vectors (2.5 \u00b5g), a pSG5 eukaryotic expression vector encoding the human AR [Hep3B cells were seeded into six-well tissue culture plates and cultured until they take possession of space of 70%. Transient transfection of human AR , extractLKB1 specific siRNA consisted of (5\u2032-GUACUUCUGUCAGCUGAUUdTdT-3\u2032 and 5\u2032-AAUCAGCUGACAGAAGUACdTdT-3\u2032) [SNU-423 cells were seeded into six-well tissue culture plates and cultured until they take possession of space of 70%. dTdT-3\u2032)  and was mRPLP0 and 18 s were used as a control in in vivo samples and in vitro respectively. The primers used for real-time PCR are in Total RNA was extracted using TRIzol Reagent  in both liver and cell samples. Reverse transcription was performed with 1.5 \u00b5g of total RNA and Reverse transcriptase kit  following the manufacturer\u2019s protocol. Quantitative PCR  was executed using Excel Taq Q-PCR Master Mix  and StratageneMx3000P . Primers used in real-time PCR were manufactured by Bionics Inc.  or Genotech . Both protein samples of livers and the cells of SNU-423 and Hep3B were extracted by using protein lysis buffer, called T-PER reagent  and quantified by Bradford assay with PRO-Measure solution . The samples were run SDS-PAGE electrophoresis on 10% polyacrylamide gels and transferred to the membrane. And the membranes were blocked with 30 mg/mL BSA100 , diluted TBS-T buffer . Primary antibodies  were ope3) at 65 \u00b0C overnight and was purified using the G-Spin DNA Extraction Kit . AR standard ChIP enrichments were quantified by PCR analysis using specific primers.To clarify whether androgen receptor binds with genes related to de novo lipogenesis, we performed chromatin immunoprecipitation (ChIP). The sample was prepared with SNU-423 cells, which were supplemented with testosterone (10 nM) and incubated for 2 h. DNA-protein cross-linking was achieved by using formaldehyde  at room temperature for 10 min. The cells were washed with cold PBS twice and resuspended by lysis buffer . Then, the sample was sonicated on ice at power 25 for 10 s pulses 10 times, using a VirSonic 100 (Virtis) sonicator. After the sonicated sample was centrifuged at 13,000 rpm for 15 min at 4 \u00b0C, chromatin (84.56 \u00b5g) was diluted in 2.5\u00d7 ChIP dilution buffer  and incubated overnight at 65 \u00b0C with anti-AR monoclonal antibody  or mouse IgG antibody as a control  and 50 \u03bcL of Dynabeads Protein A .The beads were washed 6 times with LiCl buffer , and washed briefly with TE buffer , in turn. Then, the washed sample was de-crosslinked .Data are reported as the mean \u00b1 standard deviation (SD). Differences between means were obtained by Student\u2019s Our data all suggest that NAFLD development was accentuated in conditions where the supply of androgens is limited. When AMPK-ACC signaling is generally considered the mainstream in de novo lipogenesis, testosterone modulates the signaling. Intuitively, testosterone could lead to the reduction of hepatic TG contents. We confirmed that LKB1 regulated AMPK-ACC signaling and that testosterone interacts directly with the LKB1 gene to evoke a direct AR binding. This study has increased our understanding of how testosterone acts to regulate NAFLD development and its relevance to androgen-responsive LKB1 in the male liver."}
+{"text": "The dextro-transposition of the great arteries (d-TGA) is one of the most common congenital heart diseases. To identify biological processes that could be related to the development of d-TGA, we established induced pluripotent stem cell (iPSC) lines from two patients with d-TGA and from two healthy subjects (as controls) and differentiated them into endothelial cells (iPSC-ECs). iPSC-EC transcriptome profiling and bioinformatics analysis revealed differences in the expression level of genes involved in circulatory system and animal organ development. iPSC-ECs from patients with d-TGA showed impaired ability to develop tubular structures in an in vitro capillary-like tube formation assay, and interactome studies revealed downregulation of biological processes related to Notch signaling, circulatory system development and angiogenesis, pointing to alterations in vascular structure development. Our study provides an iPSC-based cellular model to investigate the etiology of d-TGA. Congenital heart disease (CHD) represents almost one-third of all congenital diseases  and occuPitx2 and Tbx2, have been evaluated in animal models [Hspg2)-null mouse embryos present with complete TGA [Tbx2-Tgf\u03b22 pathway [d-TGA is generally reported with Heterotaxy syndrome, but the etiology and morphogenesis of TGA are still largely unknown. Nonetheless, several genes, such as l models ,7. Durinl models . During l models . Subendol models . The corlete TGA , suggestlete TGA , directlSeveral studies have used animal models of TGA to better understand disease mechanisms, and there have only been a few studies reported in humans (reviewed in ). The emIn the present study, we sought to generate a d-TGA cellular model based on the use of patient-specific iPSCs. Specifically, we generated two iPSC lines from patients with d-TGA, which were differentiated into iPSC-ECs. Several genes were identified by RNA-seq as potential players in the etiology of this congenital defect. We show that iPSC-ECs from patients with d-TGA have an impaired capacity to develop vascular networks concomitant with changes in the transcriptome, as assessed by bioinformatics analysis. Specifically, gene ontology (GO) biological processes related to embryonic morphogenesis/development, tube development and angiogenesis, together with Notch signaling pathways, were downregulated in EC derived from patients with d-TGA.CD31 and neuropilin-1 (NRP-1), a coreceptor of vascular endothelial growth factor (VEGF), at day 12, using fluorescence-activated cell sorting (FACS), which ensured an EC population. The results of the sorting analysis revealed a similar but low efficiency of EC differentiation in all four iPSC lines: FACS efficiency was 0.93 \u00b1 0.69 (Ctrl1), 2.8 \u00b1 2.08 (Ctrl2), 0.57 \u00b1 0.23 (TGA1) and 0.6 \u00b1 0.4 (TGA2). Isolated ECs showed a characteristic cobblestone morphology by brightfield microscopy  and receptor tyrosine kinase (TIE2) increased over time  and also one control iPSC line from a healthy individual (Ctrl1), which were cultured under feeder-free conditions. We used the commercially available iPSC line IMR90-4 as a second control (Ctrl2). The cells were maintained undifferentiated and displayed typical morphology A and norcroscopy C and werver time D. All iPanalyzed C. We next performed RNA-seq and bioinformatics analysis of sorted double-positive cells at day 20 of differentiation of the four iPSC-EC lines. Despite the known variability that exists between individuals ,18 and tp-value \u2264 0.05 and fold change \u2265 2.0) in iPSC-ECs from d-TGA and control lines (COL1A1), COL1A2 and laminin subunit alpha 4 (LAMA4). We also found an increase in the expression of the transcription factors SIX1 and TBX2, which have important roles in heart morphogenesis [FOXC1) with an important role in vascular development was also upregulated in iPSC-ECs from patients with d-TGA. A total of 4672 genes were identified by bioinformatics analysis, and 4471 genes had similar expression values in d-TGA and control groups. Identified genes with fewer than 150 counts were removed. We generated a volcano plot to visualize differentially expressed genes (DEG)  [JAG2) and Delta-like canonical Notch ligand 4 (DLL4). In addition, genes involved in gap junctions, such as gap junction protein alpha 4 (GJA4) and GJA5, which are necessary to establish cell communication, were also downregulated. Likewise, vascular stabilizing genes related to the ECM, such as collagen type IV alpha 1 chain (COL4A1), COL4A2 and HSPG2, were downregulated. A complete list of the upregulated and downregulated DEG is shown in Of the 139 downregulated genes in the d-TGA group, we identified several genes involved in vascular compartment and developmental processes. For example, specific endothelial markers, including  2 (KDR)  were allp-value, is shown in In agreement with the gene expression profiling, GO analysis of DEG demonstrated different biological processes associated with control and d-TGA iPSC-ECs. A list of all significantly overrepresented GO biological processes of upregulated and downregulated genes, as a function of their p-value  and the most representative are listed in As a complementary representation, we used a REVIGO treemap to reduce the number of GO terms into \u2018clusters\u2019 and \u2018superclusters\u2019 of related terms, as described , where Gp-value C. The 30These results suggest that the downregulated genes identified in iPSC-ECs from patients with d-TGA are of particular relevance for understanding the mechanisms of d-TGA in our system.We also created a treemap to group the GO biological processes, which were mostly grouped into the \u2018regulation of multicellular organismal process\u2019 and \u2018circulatory system development\u2019 superclusters D.versus controls revealed significant enrichment of genes involved in the circulatory system.Several GO biological processes were identified when the analysis was performed with up- and downregulated genes. GO analysis of genes downregulated in iPSC-ECs from patients with d-TGA versus controls  are involved in ECM organization (GO:0030198). These data suggest that the expression of different genes related to ECM is altered in iPSC-ECs from patients with d-TGA. HSPG2, a glycosylated protein component of the ECM, was downregulated in iPSC-ECs from d-TGA patients. Interestingly, Hspg2-null embryos presented with d-TGA [We studied the gene expression patterns in iPSC-ECs from patients with d-TGA, the deregulation of which may be related to the onset of disease. Two iPSC lines were successfully generated from patients with d-TGA and were differentiated into ECs. Despite the low efficiency of EC differentiation, the FACS-sorted  looping . It has  looping . Indeed,r growth . Anotherng cells . Two memrtic ECM . Differeth d-TGA , supportGJA4 have been previously related to coronary artery disease [Tbx2 and GJA4 might be implicated in the development of d-TGA.Genetic polymorphisms in  disease . This geJAG2, is directly implicated in angiogenesis [DLL4 is required for normal arterial patterning [DLL4 has an antiangiogenic effect when it competes with JAG1 [CDH5 was also found to be downregulated in iPSC-ECs from patients with d-TGA. CDH5 is a transmembrane cadherin protein required for vascular morphogenesis, as its deficiency impairs vasculogenesis in embryonic stem cells derived from embryoid bodies [KDR, which has an important role in endothelial specification and maintenance, was also observed. The activation of this receptor is critical for enhancing the proliferation and survival potential of iPSC-ECs [Regarding the downregulated genes expressed in EC from patients with d-TGA, we found several different genes involved in Notch signaling. One of these genes, ogenesis , and DLLtterning . In addiith JAG1 . Thus, dd bodies  and in md bodies . These dd bodies . The dowiPSC-ECs .At the functional level, we observed that endothelial cells derived from patients with d-TGA were unable to form capillary-like tubular networks. ECs are directly implicated in angiogenesis, which plays a crucial role during embryonic development and organogenesis . These fThe major limitation of our study is the lack of a multicellular system to simulate d-TGA in vitro. However, we have generated an in vitro model of d-TGA using iPSCs as a disease modeling approach. The gene expression differences between the ECs from control and d-TGA patients might be related to the etiology of this cardiac disease. The results presented in this work contribute to a better understanding of the mechanisms promoting the onset of d-TGA during embryonic development. Overall, our results show that ECs from patients with d-TGA display genetic differences versus control counterparts. These changes affect biological processes related to the circulatory system and to vascular development. Accordingly, ECs derived from d-TGA iPSCs could be a good cellular model for the study of this pathology. This work supports the use of patient-specific iPSC-ECs in modeling d-TGA.Oct4, Nanog, Lin 28 and Sox2 genes. Different clones were obtained and were validated using the following assays: alkaline phosphatase, surface marker expression, pluripotency gene expression, transgene silencing, proviral integration and in vitro and in vivo differentiation (data not shown).Foreskin fibroblasts from a healthy individual and a patient with d-TGA (Ctrl1 and TGA1) were obtained through ATCC and the Coriell Institute, respectively. Fibroblasts were reprogramed following the method described by Yu in 2007 , using aOct4, Sox2, Nanog and Lin28). The iPSC-TGA line TGA2 (SCVI-235) was generated from peripheral blood mononuclear cells [Ctrl2 line (IMR90-4) was purchased from WiCell Research Institute  and was reprogramed using viral transfection (ar cells  and was 2 and 21% O2 (21% O2). Cells were grown with mTeSR1 medium  and were passaged at a 1:10 ratio when ~80% confluency was reached (after about 4 days) using Accutase solution (Stemcell Technologies).All pluripotent cell lines and differentiation cultures were maintained in Matrigel  pre-coated plates at 37 \u00b0C in a Thermo Forma 370 Steri Cycle Incubator  with 5% CO\u22122 onto a Matrigel pre-coated 24-well plate (day-2) and were incubated for 2 days in mTeSR1 medium. On day 0, cells were cultured with Stemline\u00ae II Hematopoietic Stem Cell Expansion Medium  containing 10 ng/mL of Activin A , FGF-2 , BMP-4  and VEGF  for 1 day to initiate the differentiation. The following day, the medium was replaced and cells were incubated with the same concentration of FGF-2, BMP-4 and VEGF (differentiation medium) for 11 days. On day 12, FACS was performed to isolate endothelial cells.Endothelial cell differentiation was induced following the protocol described by Prasain et al. in 2014 . BrieflyCD31; PECAM-1, Alexa Fluor 488-conjugated, BD-Pharmingen, San Diego, CA, USA) and Neuropilin-1 . Double-positive CD31+/NRP-1+ cells were isolated by FACS and were expanded in collagen-coated T25 flasks until confluence [TM . Experiments were performed from day 19 of differentiation and cells were maintained in culture for no more than 5 passages.Cells were detached by trypsinization and filtered through a 40-\u00b5m filter to form a single cell suspension. Cells were resuspended in 130 \u00b5L of PBS, supplemented with 1% fetal bovine serum and were incubated with anti-human platelet/endothelial cell adhesion molecule-1  with a 10\u00d7 magnification and were analyzed with WimTube online software . Five images from independent experiments were analyzed for each iPSC-EC line. iPSC-ECs were plated onto Matrigel-coated wells to measure tube formation, as described . BrieflyGAPDH) was used as housekeeping control. Reactions were performed in triplicate.RNA was isolated from samples on different days of EC differentiation  using the RNeasy Plus kit . cDNA was produced using a High-Capacity RNA-to-cDNA Kit , and real-time PCR (qPCR) was performed using the LightCycler 480 SYBR Green I Master Kit  on a ViiA 7 Real-Time PCR System . Primers were provided by Condalab  and glyceraldehyde-3-phosphate dehydrogenase . Briefly, 300 ng of total RNA was used for ribosomal RNA depletion. Then, ribosomal-depleted RNA was fragmented for 4.5 min at 94 \u00b0C. The remaining steps of the library preparation were followed according to the manufacturer\u2019s instructions.Libraries were analyzed on an Agilent Technologies 2100 Bioanalyzer system, using the Agilent DNA 1000 chip to estimate the quantity and validate the size distribution, and were then quantified by qPCR using the KAPA Library Quantification Kit KK4835 . FASTQ files were treated with Cutadapt and aligned with Kallisto to obtain the count table for each gene. Differential expression analysis of RNA-seq expression profiles was performed with the EdgeR package . GO enriAnalysis was performed as described . Complet\u00ae software . Differences were considered statistically significant at p < 0.05, with a 95% confidence interval.Results are represented as the mean \u00b1 standard deviation (SD). Comparisons between Ctrl and TGA groups were performed with the non-parametric Mann\u2013Whitney U test, one-way ANOVA and necessary post hoc analysis. Analyses were conducted with GraphPad Prism 8"}
+{"text": "Accompanying co-morbidities in patients with ovarian cancer are of major relevance for scheduling debulking surgery, especially in the anesthesiological consultations. Aim of this study was to evaluate the impact of co-morbidities and patient characteristics on postoperative complications.Patients undergoing maximal cytoreductive surgery were prospectively enrolled from October 2015 to January 2017. Various variables were recorded, such as the Charlson comorbidity index, Eastern cooperative oncology group scale of performance status (ECOG PS) and the American society of anesthesiologists physical status classification system (ASA PS). Surgical complications were graded using the Clavien\u2013Dindo criteria. Logistic regression models were used to analyze risk factors for severe postoperative complications.p\u2009=\u20090.01), body mass index (BMI)\u2009>\u200925\u00a0kg/m2  along with the use of intraoperative norepinephrine\u2009>\u20090.11\u00a0\u00b5g/kg/min  and intraoperative fresh frozen plasma (FFP)\u2009>\u200917\u00a0units  appeared as significant predictors of severe postoperative complications.Of 106 enrolled patients, 19 (17.9%) developed severe postoperative complications grade\u2009\u2265\u2009IIIb according to Clavien\u2013Dindo criteria. In the multivariable regression analysis impaired (ECOG PS)\u2009>\u20091 (odds ratio OR) 13.34, 95% confidence interval (CI) 1.74\u2013102.30, 2), are highly predictive factors for severe postoperative complications. The analysis of intraoperative data showed that the need for more than\u2009>\u20090.11\u00a0\u00b5g/kg/min of norepinephrine and transfusions of FFPs more than 17\u00a0units were strongly associated with severe postoperative complications.We demonstrated that neither the presence of a certain comorbidity nor the summation of the co-morbidities were associated with adverse outcome. Patient characteristics, such as ECOG PS\u2009>\u20091 and obesity (BMI\u2009>\u200925\u00a0kg/mThe online version contains supplementary material available at 10.1007/s00404-021-06116-5. Complete cytoreduction is one of the main prognostic factors for patients with ovarian cancer \u20133. To acThe aim of this study was to evaluate potential predictive markers for severe postoperative complications in ovarian cancer surgery, such as co-morbidities and further clinical characteristics.This analysis is part of the prospective study named \u201cRISC-GYN\u2014trial\u201d. The study was designed to explore predictive markers for severe postoperative complications in gynecologic cancer patients. Out of this cohort with gynecological cancer patients, we selected 155 ovarian cancer patients as the largest entity within the RISC-GYN\u2014trial. We later excluded patients with recurrent disease. See Fig.\u00a0The Charlson comorbidity index was calculated for each patient as previously published by Charlson et al. . By addiInformation on performed operative procedures, perioperative anesthesiologic parameters and tumor dissemination pattern was documented intraoperatively. The intraoperative mapping of ovarian cancer (IMO) was applied for exact tumor documentation .Each patient was visited systematically in a daily schedule until the end of their hospital stay and was followed up via phone after 3\u00a0months post surgery. Our primary outcome of interest was 30-day postoperative complications Clavien\u2013Dindo classification grade IIIb, IV and V. Clavien\u2013Dindo classification grade IIIb complications require an intervention under general anesthesia . See SupWhere appropriate the Fisher\u2019s exact test for dichotomous variables, Kendall\u2019s tau b for ordinal variables, the chi test for nominal variables\u00a0and Kruskal\u2013Wallis test or Mann\u2013Whitney test for continuous variables were performed to evaluate comparisons between groups.pin\u2009=\u20090.05 and pout\u2009=\u20090.10. Excluded from the multivariable analyses were cases with missing values. For statistical analysis, IBM\u00ae SPSS\u00ae Statistics 25  was used and statistical significance was defined as p\u2009<\u20090.05 without alteration for multiple comparisons.To assess the predictive accuracy of continuous variables for distinguishing patients with severe postoperative complications from those without severe postoperative complications and to determine cut-offs, receiver-operator characteristics (ROC) curve analyses were used see Fig.\u00a0. LogistiA total of 106 women undergoing surgery for ovarian cancer were enrolled in the study. Baseline characteristics of the study cohort are presented in Table n\u2009=\u200914, 13%), postoperative pleural effusions , anastomotic insufficiency , wound dehiscence , peritonitis , and thromboembolic events . Table n\u2009=\u200934, 32%), polyneuropathy , chronic pulmonary disease  and diabetes mellitus type I and type II . Cardiovascular disease  was defined as current or condition after myocardial infarction, heart failure, peripheral vascular disease, cerebrovascular disease, cardiac arrhythmias and atrial fibrillation. See Table Overall, 19 patients (17.9%) experienced complications Clavien\u2013Dindo Classification grade\u2009\u2265\u2009IIIb and one of them  died within 30\u00a0days of surgery. The most common complications were urinary tract infections , a higher rate of polypharmacy , diabetes , compared to patients without or with less severe complications. They showed significantly more often hypokalemia , hypoalbuminemia  and a high level of CA 125 (>\u2009500\u00a0U/ml) . Their ECOG PS was more often impaired\u00a0 age as a continuous variable  and age\u2009\u2265\u200970\u00a0years  showed no association with severe postoperative complications.The patients with serious postoperative complications had significantly more co-morbidities  and , respectively.An ASA PS\u2009>\u20092 and ECOG PS\u2009>\u20091 were associated with severe complications . The age-adjusted Charlson comorbidity index showed significance at a cut-off point of\u2009>\u20092 .Univariate logistic regression showed further that an increased number of co-morbidities (Charlson comorbidity index\u2009\u2265\u20091) was significantly associated with severe postoperative complications  and diabetes , bowel obstruction symptoms  and the simultaneous intake of more than five medications  were significantly related in univariate logistic regression, whereas hypertension was not. Liver disease, chronic pulmonary disease and renal disease did not show correlation to severe postoperative complications. Hypokalemia (<\u20093.7\u00a0mmol/l) , a decreased INR (\u2264\u20090.9)  and hyperbilirubinemia (>\u20090.46\u00a0mg/dl)  were associated with severe postoperative complications.Cardiovascular disease  and obese patients with a BMI above 30\u00a0kg/m2 were up to 9 times more likely to develop severe postoperative complications .Overweight patients with a BMI\u2009>\u200925\u201330\u00a0kg/mp\u2009=\u20090.02) showed the greatest correlation with severe postoperative complications. Overweight and obesity  were highly predictive as well. Furthermore, the Charlson comorbidity index  was an independent predictive parameter. The age-adjusted Charlson comorbidity index and all the assessed co-morbidities lost significance in multivariate analysis. See Table All the preoperative variables were included in the stepwise logistic regression. ECOG PS\u2009>\u20091 , the small intestine , upper abdominal surgery  and pelvic and/or paraaortical lymphonodectomy . In univariate analysis, most of the surgical procedures, prolonged duration of surgery, intraoperative hypothermia (<\u200936\u00a0\u00b0C) and most of the tumor-related characteristics did not show significant correlation with complications.The surgical procedures of special interest included bowel resection with resection of the large intestine (p\u2009=\u20090.05), the use of fresh frozen plasma  and a level of CA 125\u2009>\u2009500\u00a0U/ml  showed significant association.Only the use of more than 0.11\u00a0\u00b5g/kg/min norepinephrine  and the pelvic and/or para-aortic lymphadenectomy  remained significantly associated with severe postoperative complications.The intraoperative and tumor-related parameters were also analyzed in a stepwise logistic regression. Only the use of more than 17 intraoperative fresh frozen plasma units , ECOG PS\u2009>\u20091 , the use of more than\u2009>\u20090.11\u00a0\u00b5g/kg/min norepinephrine  and the use of more than 17 intraoperative FFP units  predictive for severe postoperative complications.Ultimately, to all significant factors from the preoperative analysis, the intraoperative and tumor-related parameters were added into stepwise logistic regression. Table Several studies report an association between co-morbidities and clinical outcome but most of them are performed retrospectively and heterogeneous patient populations limit their interpretation , 22. TheWe screened systematically all co-morbidities and several preoperatively assessed patient-related characteristics, as well as intraoperative anaesthesiological, surgical and tumor-related parameters in this prospective study to assess their impact on 30\u00a0days\u2019 postoperative complications in ovarian cancer patients. In the first part of the data analysis, we demonstrated that preoperative patient-related characteristics, such as ECOG PS\u2009>\u20091, obesity and the Charlson comorbidity index, are predictive factors for postoperative complications. In the second part, the analysis of intraoperative data showed the performance of lymphadenectomy and the high need for fresh frozen plasma transfusions to be strongly associated with severe postoperative complications.2, high need of intraoperative FFP and norepinephrine are associated with severe postoperative complications.The combined evaluation of preoperative and intraoperative factors showed ECOG PS\u2009>\u20091, overweight and obesity/a BMI\u2009>\u200925\u00a0kg/mThe age of the patients showed no association with complications, not even for patients above the age of 70\u00a0year. Other studies showed that older patients are at increased risk of surgical morbidity, but if they are able to tolerate aggressive surgical management, the benefit was equivalent to that of younger women . In contPrevious studies have identified the ASA PS and ECOG PS as good tools for outcome prediction in cancer patients , 23, 26.Several publications focusing on ovarian cancer showed obesity as a highly predictive factor for postoperative complications , 26\u201332. In our analysis of intraoperative data, we intentionally did not group the surgical interventions as other studies did . To examIn our study, the use of high levels of norepinephrine and FFP correlated significantly with postoperative complications. The effect of vasoactive medication on gastrointestinal oxygen supply and microcirculatory blood flow is controversially discussed in several studies \u201337. The In this study, we screened all accompanying co-morbidities in patients with ovarian cancer systematically. Neither the summation of the co-morbidities assessed by the Charlson comorbidity index nor the presence of a certain comorbidity was associated with postoperative complications in the multivariate analysis of pre- and intraoperative parameters.Our findings imply that the patient characteristics, such as ECOG performance status and BMI, could have greater effect on complications than the co-morbidities and the complex surgical interventions themselves.There is a limitation noted for this study, as we included patients with different tumor stages and analyzed a heterogenous set of data. As our results are based on regression analysis, we cannot simply state causation, especially as the individual variables are influenced by each other. However, our findings contribute to a clearer picture of patients who will benefit from surgery.The main strength of our study is, that it is a prospective study which examined predictive factors for postoperative complications as the primary endpoint. Using the Clavien\u2013Dindo classification to grade postoperative complications made our results more objective as well as comparable and reproducible for future research. The homogeneous and very high quality of surgical treatment as well as daily visits of each patient lead to further improvement of reliability and validity of the acquired data.Surgeons should consider performance status rather than the sum of co-morbidities in future risk prediction. In this study, we identified potential parameters that can be preoperatively easily assessed and are feasible to collect in clinical routine. ECOG PS and BMI should be given more attention in clinical decision-making, treatment planning and patient counseling. A high intraoperative use of norepinephrine and FFP during cytoreductive surgery should lead to interdisciplinary communication between surgeons and anesthesiologists to give more awareness of the vulnerability of patients for complications.Supplementary file1 (DOCX 16 kb)Below is the link to the electronic supplementary material."}
+{"text": "Local recurrence is common after curative resections for rectal cancer. Surgical intervention is among the best treatment choices. However, achieving a negative resection margin often requires extensive pelvic organ resections; thus, the postoperative complication rate is quite high. Recent studies have reported that the inflammatory index could predict postoperative complications. This study aimed to validate the correlation between clinical factors, including inflammatory markers, and severe complications after surgery for local recurrent rectal cancer.This retrospective study included 99 patients that underwent radical resections for local recurrences of rectal cancer. Postoperative complications were graded according to the Clavien-Dindo classification. Grades \u22653 were defined as severe complications. Risk factors for severe complications were identified with univariate and multivariate logistic regression models and assessed with receiver-operating characteristic curves.p\u2009=\u20090.02) and a blood loss \u22652850\u2009mL  were associated with a significantly higher incidence of severe postoperative complications.Severe postoperative complications occurred in 38 patients (38.4%). Analyses of correlations between inflammatory markers and severe postoperative complications revealed that the strongest correlation was found between the prognostic nutrition index and severe postoperative complications. The receiver-operating characteristic analysis showed that the optimal prognostic nutrition index cut-off value was 42.2 . In univariate and multivariate analyses, a prognostic nutrition index \u226444.2 (Odds ratio: 3.007, 95%CI:1.171\u20138.255, We found that a low preoperative prognostic nutrition index and excessive intraoperative blood loss were risk factors for severe complications after surgery for local recurrent rectal cancer.The online version contains supplementary material available at 10.1186/s12885-021-08160-5. Colorectal cancer is one of the most common cancers in the world . The 5-yLRRC can be cured with radical resection. The reported 5-year survival rate is 43 to 70% after a curative resection for LRRC . ConsequRecent studies have shown that inflammatory markers, such as the neutrophil/lymphocyte ratio (NLR) and the platelet/lymphocyte ratio (PLR), were useful for predicting postoperative complications and prognosis , 14. TheThe purpose of this study was to clarify the correlation between severe postoperative complications and various clinical factors, including inflammatory markers, in LRRC.This retrospective cohort study was performed in Osaka University Hospital. We collected information for patients that had undergone curative resections for LRRC between January 2000 and January 2020. A total of 99 patients were eligible for this study.We acquired information on patient characteristics, including age at surgery, sex, body mass index (BMI), and the preoperative physical state, assessed with the American Society of Anesthesiologists (ASA) physical status classification. In addition, we examined the location and TNM stage of the primary tumor , the postoperative adjuvant therapy given after the initial treatment, and the preoperative treatment for LRRC surgery. We also included data on intraoperative details, including the surgical procedures, surgical approaches, combined resected organs, operation time, blood loss volume, and residual tumor (R status). We assessed the severity of postoperative complications with the Clavien-Dindo (CD) classification. Severe postoperative complications were defined as CD grade\u2009\u2265\u20093.The latest laboratory data processed before the LRRC surgery was collected, including the complete blood count and the serum albumin and CRP levels. Serum CRP levels under 0.04\u2009mg/L were below the detection limit of our measuring instrument; therefore, values under 0.04\u2009mg/L were set to 0.04\u2009mg/L for this analysis. These data were used to calculate the inflammatory markers, NLR, PLR, lymphocyte/CRP ratio (LCR), CAR, and PNI.All categorical data were presented as number of cases and percentages, while continuous data were shown as median and interquartile range (IQR). We performed logistic regression analyses to assess correlations between the incidence of severe postoperative complications and each inflammatory marker. We then created receiver operating characteristic (ROC) curves and compared areas under the ROC curves to determine the strongest effective marker of severe postoperative complications. We performed multiple logistic regression analyses to assess the correlation between severe postoperative complications and clinical factors.p-values <\u20090.05 were considered statistically significant. We calculated the exact 95% confidence intervals (95% CIs) for absolute differences and odds ratios (ORs).All data were processed and analyzed with JMP Pro 14.0 . Two-tailed This study was performed in accordance with the Declaration of Helsinki . The study protocol was approved by the Ethics Committee of Osaka University Hospital. All study participants provided written informed consent.2 [IQR 20.2\u201324.1]. The majority of the patients were class 2 of preoperative physical state . A total of 63 patients (63.6%) received neo-adjuvant therapy for LRRC. More than a half of the patients  had chemoradiotherapy, whereas smaller numbers had chemotherapy  or radiotherapy alone . About primary tumor characteristics, more than 40% of the primary tumors were located in the lower rectum . Regarding the T stage of the primary tumors, the majority of patients was T3 or T4 . About half of the patients had lymph node metastasis in the primary cancers . Adjuvant chemotherapy for the primary cancer was given to more than half of the patients .Table\u00a0Table\u00a0n\u2009=\u200979; 79.8%) underwent a laparotomy and the rest of 20 patients (20.2%) underwent laparoscopic surgery. Regarding the type of surgery, the most common procedure was a tumorectomy or lateral lymph node dissection  and a TPE was performed in 22 patients (22.2%). More than 40% of the patients  underwent a combined sacral resection. The median operation time was 680\u2009min [IQR 438\u2013871], and the median intraoperative blood loss was 2850\u2009mL [IQR 530\u20135240]. In histopathological outcome, the complete resection rate (R0) was 93.9% (n\u2009=\u200993).Table\u00a0The incidence of postoperative complications is shown in Table\u00a0p-value for each inflammatory marker are shown in each panel. The PNI showed the strongest correlation with severe postoperative complications in LRRC. The PNI cutoff value for indicating severe complications was 44.2, and it showed a sensitivity of 0.790 and a specificity of 0.508.To identify correlations between inflammatory markers and severe postoperative complications in LRRC, we performed univariate analyses. Then, we created ROC curves to analyze the sensitivity and specificity of each inflammatory marker that was correlated to severe postoperative complications Fig.\u00a0. The arep\u2009=\u20090.02) and blood loss \u22652850\u2009mL  were independent risk factors . As a result, the NLR and PLR might be less sensitive to severe postoperative complications than expected. Another potential explanation might be the limited accuracy of our blood test equipment. Because we could not assess CRP levels below 0.04, CRP levels less than 0.04 were treated as 0.04 in this study. Therefore, we could not accurately assess the CAR and LCR values, which required the CRP level.The relationship between blood loss and postoperative complications was previously reported in colorectal cancer surgery , 21. HeaSome previous studies have reported that preoperative nutritional interventions were effective for postoperative outcomes in various types of cancer. Indeed, preoperative exercise and nutritional support improved the postoperative outcome in patients with gastric cancer . DespiteWe previously reported that laparoscopic surgery was safe and useful for LRRC . The magThis study had some limitations. First, the study was retrospective, and data were from a single center. Second, the cohort was relatively small. Although 99 patients represented a relatively large cohort in clinical research for LRRC, it was insufficient to clarify the risk factors for postoperative complications.In conclusion, we found that a PNI <\u200944.2 and blood loss \u22652850\u2009mL were independent risk factors for severe complications after LRRC surgery. Preoperative interventions that improve the nutritional status and an approach that reduces surgical invasiveness could potentially reduce the risk of severe complications after LRRC surgery.Additional file 1: Supplemental Table\u00a01. Primary tumor characteristics of the patients in this study."}
+{"text": "In the past decade(s), fluorescence microscopy and laser scanning confocal microscopy (LSCM) have been widely employed to investigate biological and biomimetic systems for pharmaceutical applications, to determine the localization of drugs in tissues or entire organisms or the extent of their cellular uptake (in vitro). However, the diffraction limit of light, which limits the resolution to hundreds of nanometers, has for long time restricted the extent and quality of information and insight achievable through these techniques. The advent of super-resolution microscopic techniques, recognized with the 2014 Nobel prize in Chemistry, revolutionized the field thanks to the possibility to achieve nanometric resolution, i.e., the typical scale length of chemical and biological phenomena. Since then, fluorescence microscopy-related techniques have acquired renewed interest for the scientific community, both from the perspective of instrument/techniques development and from the perspective of the advanced scientific applications. In this contribution we will review the application of these techniques to the field of drug delivery, discussing how the latest advancements of static and dynamic methodologies have tremendously expanded the experimental opportunities for the characterization of drug delivery systems and for the understanding of their behaviour in biologically relevant environments. The development of nanomaterials for the delivery and controlled release of drugs to a selected, specific biological target has been, for many years, one of the core areas of nanomedicine research. Indeed, the design and preparation of smart nanocarriers\u2014with encoded targeting and/or controlled release abilities, multifunctional therapeutic or combined therapeutic/diagnostic properties, and responsivity to diverse stimuli\u2014have reached, over the years, unpredictable achievements . SignifiIn this framework, fluorescence microscopy and laser scanning confocal microscopy (LSCM) are key experimental techniques to unravel the behavior of nanomaterials designed for pharmaceutical applications in biological environments ,11,12,13In addition, aside from imaging techniques, which provide the ability to unravel the localization of DDSs in complex environments, several fluorescence microscopy-related techniques have been developed and refined over the years, thus allowing for informational gain related to the dynamics of DDSs . Fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and particle tracking (PT) can provide  information on the dynamics of DDSs, or on the modification of the typical dynamics of biological environments in response to the interaction with a DDS. Crucial information on DDS characteristics and behaviors in a complex biological environment can be obtained through these techniques, as the internalization mode of a nanocarrier inside a cell lumen , its adhesion to a biological interface, and its interaction with relevant biomolecules present within biological fluids ,15,16,17Despite the extensive application of LSCM in the characterization of biological systems and nanostructured DDSs, a clear limitation is represented by the achievable spatial resolution, which is far from that of electron microscopes. Indeed, typical sizes of nanostructured DDSs are within the range of a few\u2013a few tens or hundreds of nanometers, which are also the typical length scales of DDS interactions with the surrounding environment, while the resolution limits of LSCM are 200\u2013300 nm in the xy plane and 500\u2013700 nm in the axial direction . Due to this inherent physical limit imposed by the diffraction limit of light, the information obtained through LSCM on the characteristics of nanostructured DDSs, as well as on their impact on subcellular processes or interaction with biological interfaces, is limited.In recent years, different methods have been developed to break the physical diffraction limit of light, approaching the typical nanometric resolution of electron microscopy. From the recognition of the potential groundbreaking impact of super-resolution imaging, with the 2014 Nobel prize in Chemistry, different nanoscopy techniques have been implemented on commercial microscopes, and are constantly refined to expand their applicability. Driven by this new hype around super-resolution microscopy, a general renewed interest has grown on advanced fluorescence microscopy techniques, which now offer multiple options and new opportunities for the characterization of nanostructured objects as DDSs, and of their behaviors  in biological environments, on a nanometric length scale.In this review, we summarize the major recent advancements of fluorescence microscopy-related techniques, in view of their impact in the field of drug delivery. Specifically, in The main application of advanced fluorescence microscopy-based techniques to the investigation of DDSs is the determination of the localization of DDSs in vitro and in vivo, which is a key issue, for instance, to: (i) understand the behavior of DDSs with biological interfaces/barriers and in biological media; (ii) evaluate the degree of cell uptake and understand cell uptake routes in vitro; and (iii) determine the extent of DDS accumulation in selected tissues. In these respects, the main advantage represented by super-resolution techniques is the possibility to accurately determine the localization of DDSs in complex biological samples. Clearly, together with opportunities, several challenges arise. To mention a few: some super-resolution techniques necessitate the use of powerful laser sources , which on one hand requires a tailored design of optimized photostable fluorescent probes, while on the other hand, might determine the photodamage and phototoxicity of biological samples in cases of long exposure ; biologiIn the latest years, the advent of super-resolution microscopy (SRM) has represented a game-changer in the characterization of the behavior of DDSs in biological systems\u2014and more in general, in biomedical research\u2014holding the promise to combine the inherent advantages of confocal microscopy  with an extremely high resolution, thus approaching the limits of electron microscopy. A thorough description of the different SRM techniques is beyond the scope of this review ; STED imaging, first conceptualized in 1994 by Hell et al. , enablesA different approach to achieving the super-resolution adopted in super-resolved, single-molecule localization microscopy is based on the localization of the fluorophores\u2019 position under diffraction limit accuracy, which is completed by determining the centroid of the emitting spot. In particular, as briefly schematized in \u00ae) targeting cancer cell surface biomarkers) on functionalized liposomes [From an applicative perspective, all SRM techniques represent powerful tools in cellular biology and pharmaceutical application. Each of the techniques provides direct visualization of engineered DDSs in vitro and in vivo in cells, tissues, and living organisms, and thus, direct proof of their efficacy in reaching their biological target. However, they can also represent valuable tools in characterizing complex, nanostructured DDSs; complementing the structural, colloidal, and interfacial information obtained through common characterization techniques applied to nanomaterials\u2014such as scattering , surface techniques , and electron microscopy. For instance, STORM microscopy has been recently applied to investigate the formation of the protein corona coating silica nanoparticles, allowing for the quantification of the dynamic inhomogeneities in the protein corona layer  and thusiposomes , showingAside from these examples where SRM is applied to characterize the DDSs themselves, the key contribution of SRM to drug delivery research is represented by the possibility to achieve a direct visualization and the sub-diffraction, nanometric localization of DDSs in biological environments. In this respect, a general issue in SRM is in the choice of suitable fluorescent probes to appropriately label both the nanocarriers/active principles and cellular/subcellular compartments. This issue also applies to fluorescence microscopy; however, in super-resolution techniques, the photophysical properties of the dyes, such as photostability, brightness, and the ability to control the ON/OFF switch of excited states, are determinant factors in the formation of super-resolved images, themselves. Therefore, in the latest years, researchers have focused on synthesizing/developing suitable fluorescent probes, especially for the appropriate labeling of complex cellular environments ,43,44,45A relevant contribution of super-resolution microscopy to drug delivery research is the potential ability to localize a DDS inside cells with high spatial resolution, thus allowing not only for the evaluation of its cell uptake and cell uptake extent, but also its proximity to\u2014and, therefore, its interaction with\u2014specific cellular compartments. In this respect, super-resolution techniques have been applied to understand the endocytic pathways of nanocarriers. For instance, the internalization of cancer-derived exosomes in HeLa cells was detected using PALM/STORM imaging, by revealing the colocalization inside lysosomes , while tSuper-resolution techniques have also been successfully applied to the investigation of the interaction between nanocarriers and DDSs with relevant biological barriers, such as the dermal barrier and the blood\u2013brain barrier. Specifically, Brewer et al. , by applAs already mentioned in the Concerning thin samples, total internal reflection fluorescence (TIRF) microscopy is characterized by a relatively simple experimental setup\u2014allowing for thin samples to reach a high resolution close to that of STED while applying a localized illumination with minimum time and light power exposure\u2014making it particularly suitable for live-cell imaging. As briefly schematized in Concerning thick samples, LSCM and super-resolution microscopies are generally limited to 60\u201380 \u00b5m of depth. Thick samples, as tissue slices, can be investigated through light-sheet-based fluorescent microscopy (LSFM) . The LSFAs reviewed in the previous section, thanks to the advancements of fluorescence microscopy, it is now possible to determine the localization of drug delivery with nanometric resolution. This undoubtedly represents an opportunity to understand the fate of DDSs in living organisms. Another opportunity offered by fluorescence microscopy and laser scanning confocal microscopy-related techniques is to determine not only the localization of nanosystems or drug delivery systems but also their dynamics. Techniques such as particle tracking (PT), fluorescence correlation spectroscopy (FCS), and fluorescence recovery after photobleaching (FRAP), which basic principles are summarized in We consider three main fluorescence microscopy-related techniques as possible options to investigate drug delivery systems from different perspectives. Particle tracking (PT), fluorescence recovery after photobleaching (FRAP), and fluorescence correlation spectroscopy (FCS) are characterized by two main differences: (i) they monitor dynamics and diffusion in different ways  is related to purely Brownian motion, where if the dependence of the MSD on time is t\u03b1 with \u03b1 < 1, then subdiffusive/cage/crowding effects occur, whereas with \u03b1 > 1, active transport effects are present [The PT technique is based on the acquisition/analysis of multiple images of fluorescently labeled diffusing species, which are then analyzed to determine the trajectories of each diffusing species, and therefore analyze their dynamic behavior. In particular, by evaluating the displacement between the particle positions, it is possible to calculate the mean squared displacement (MSD) of particles linked to their diffusion coefficient. The dependence of the MSD on time provides information about the diffusion modes of the particles. A linear dependence of the MSD on time  inside the sample through the employment of a high-intensity laser beam. A series of images is then acquired at normal illumination intensity to monitor the rate and extent of fluorescence intensity recovery inside the ROI. Specifically, from the rate of fluorescence intensity recovery (which is due to the replacement of the photobleached fluorescent dyes with active fluorescent species diffusing from the neighboring areas), the diffusion coefficient of the fluorescent species can be inferred; from the percentage of recovery compared to the theoretical fluorescence recovery, the mobile fraction of the probe is discriminated from the immobile fraction  . The expIn the latest years, fluorescence microscopy-related techniques have exhibited continuous developments and progresses, both from technical and applicative perspectives, holding the promise to provide unprecedented tools for drug delivery research. In this review, we have revised the major fluorescence microscopy-related experimental techniques available for the characterization of drug delivery systems from static and dynamic points of view in different media, with a particular focus on the investigation within biological environments and in vivo. Indeed, the opportunities provided by fluorescence microscopy-related techniques to disentangle scientific issues typical of drug delivery research  are countless and exponentially growing, allowing for the expectation that in the next few years the development of completely new tools and protocols will truly advance drug delivery research."}
+{"text": "Congenital epidermoid cysts are slow-growing, mass lesions caused by the abnormal inclusion of neuroectodermal tissue within the developing central nervous system. Subtotal excision of epidermoid cysts increases the risk of early recurrence of clinical signs. A 4-year-old female spayed boxer was presented with a 4-month history of ambulatory paraparesis and proprioceptive ataxia. Neurological examination localized a T3-L3 myelopathy. MRI revealed a T1 iso- to hypointense, T2 and FLAIR hyperintense, rim-enhancing mass at the level of the T9-T10 vertebrae resulting in extradural compression of the spinal cord. This was histopathologically confirmed as an extradural epidermoid cyst following subtotal excision. MRI performed 2 months post-operatively revealed a significant decrease of the lesion volume. The dog was neurologically normal following the surgery however re-presented 28 months later with recurrence of clinical signs. A 28-month post-operative MRI revealed substantial enlargement of the epidermoid cyst. The dog was subsequently taken for repeat decompressive surgery. At 6 months from the repeat surgery, the dog was neurologically static with mild proprioceptive deficits. The case report highlights the clinical and MRI features of a recurrent extradural spinal epidermoid cyst treated by subtotal excision. Congl in one , 17\u201319.MRI is the modality of choice for diagnosis and surgical planning of epidermoid cysts , 24. TypIn people, total surgical excision is the gold standard treatment for epidermoid cysts , 24. SubA 4-year-old female spayed boxer was referred to the Purdue University Veterinary Hospital for a 4-month history of progressive paraparesis and pelvic limb ataxia. Neurologic examination revealed ambulatory paraparesis with severe proprioceptive ataxia and no associated pain on spinal palpation. Based on these findings, a T3-L3 myelopathy was localized. The patient was otherwise healthy on systemic screening .On MRI, a well-defined irregularly-shaped extradural mass with approximate height of 1.2 cm and craniocaudal length of 1.4 cm was present in the right lateral aspect of the spinal canal at the level of the T9-T10 intervertebral disc space, filling ~60% of the cross-sectional area with associated marked spinal cord compression .Cerebrospinal fluid (CSF) was obtained from the cerebellomedullary cistern. Analysis revealed protein-cytological dissociation (protein \u221270 mg/dl (reference range 0\u201335 mg/dL), 655 red blood cells/\u03bcL and 2 nucleated cells/\u03bcL). Cytological evaluation demonstrated 6 large mononuclear cells and 3 non-degenerative neutrophils per high powered field with normal cytologic characteristics.in situ, though the spinal cord was adequately decompressed. Samples of the lesion were submitted for histopathology. No intra-operative complications were encountered.Following CSF collection, a T9-T10 right-sided hemilaminectomy and surgical debulking was performed. Dissection of the epaxial muscles revealed an irregular, firm, white fibrous mass adhered to the base of the T10 spinous process extending cranially to the T9-T10 articular facet. The mass extended into the spinal canal causing severe extradural spinal cord compression from caudal T9 to the mid aspect of T10. No overt capsule was visualized. Due to the firm adherence of lesion to the adjacent bone and poor visualization of the ventral extent of the mass into the T10 vertebral body, gross abnormal tissue was left Histologically, the surgical biopsy specimens included stacks of orthokeratotic lamellar keratin and a few 2-mm-long segments of keratinizing stratified squamous epithelium supported by fibrous tissue without adnexa , 4. The In addition to routine postoperative analgesia, the patient was placed on 1 mg/kg/day of prednisone. Daily neurological evaluations were performed over the 5-day period of hospitalization. The patient was ambulatory at time of discharge with improved paraparesis and moderate proprioceptive ataxia.A 2-month post-operative MRI revealed a decrease in size of the epidermoid cyst  in the spinal canal at the level of the T9-T10 intervertebral disc space with minimal spinal cord compression  which occupied ~70\u201380% of the right lateral to right dorsolateral aspect of the T9-T10 spinal canal. This resulted in severe extradural spinal cord compression with progressive extension and bony resorption of the right dorsal aspect of the cranial T10 vertebral body (At 28-months following the initial surgery, a repeat decompressive surgery was performed. There was extensive, highly vascular fibrous scar tissue overlying the T8-T11 vertebrae. The previous T9-T10 hemilaminectomy site was challenging to visualize. A right hemilaminectomy was performed at T11-12 to visualize normal spinal cord with subsequent cranial extension to the level of T8-T9. Fibrous adhesions at the previous T9-T10 hemilaminectomy window were sharply dissected with an #11 blade to expose spinal cord. The primary mass was a pale gray opalescent structure. Surrounding the mass at the cranial and caudal margins and extending into the T10 vertebral body was dark brown to black granular material, suspected to be keratin or necrotic epithelial material. The mass and granular material were debulked including the extension into the dorsal aspect of T10 vertebral body. No grossly visible abnormal tissue remained, and the spinal cord was adequately decompressed prior to routine closure of the surgical site. Samples of the lesion were submitted for histopathology with similar findings as for the original biopsy specimen and a diagnosis of epidermoid cyst. No intra-operative complications were encountered. The patient was discharged 4 days post-operatively and was strongly ambulatory with mild proprioceptive ataxia. The owner was contacted 6 months after the second surgery and reported that the patient was neurologically static; the dog remained strongly ambulatory with mild proprioceptive ataxia.This case report highlights the clinical and imaging features of repeat subtotal resection in a recurrent intraspinal extradural epidermoid cyst in a dog. Surgical excision is considered the gold standard of treatment since epidermoid cysts are neither responsive to radiation nor chemotherapy , 27. TheIn the dog reported here, complete resection was not performed in either surgery. However, the extradural location facilitated sufficient decompression of the spinal cord and associated clinical improvement. Following both surgeries, the patient showed a significant improvement immediately post-operatively. Relapse of clinical signs secondary to substantial cyst regrowth occurred 28 months after the first surgery with no deterioration of clinical status noted at 6 months after the second subtotal resection. A single case report describes surgical excision of an extradural epidermoid cyst in a boxer which was minimally affected 7 months post-operatively, but longer-term follow-up was not reported . There aA reported risk of subtotal excision is aseptic meningitis secondary to rupture of cystic contents. Free fatty acids and cholesterol accumulate within the cavity due to inflammation associated with necrosis of the degenerating squamous cells , 22. FolAlthough congenital in origin, due to their slow-growing nature, epidermoid cysts are often asymptomatic in their early stages . In peopOn MRI, epidermoid cysts show variable signal intensity based on different components of the tumor . TypicalAnother notable change on the follow-up scans was the presence of a focal intramedullary perilesional T2-hyperintensity. In intramedullary epidermoid cysts, perilesional spinal cord changes have been attributed to leakage of cystic content resulting in inflammation and gliosis along the margin of the lesion . Given tIn addition to the changes within the spinal cord noted on imaging, our patient demonstrated osseous changes in the region of the mass with overlapping characteristics to an extradural epidermoid cyst previously reported in another boxer . Both paIn summary, we report the surgical removal of a recurrent intraspinal extradural epidermoid cyst in a dog. MRI is the modality of choice for diagnosis and surgical planning of the lesion. Although signal intensity on T1W images can vary, an epidermoid cyst should be considered as a differential diagnosis for a T2 and FLAIR hyperintense, peripherally contrast-enhancing mass lesion is observed on MRI in a young to middle-aged canine. Despite subtotal excision, there was a long interval before relapse of clinical signs and improvement with re-operation. This case supports that dogs with extradural epidermoid cysts have a better outcome than previously reported dogs with intramedullary epidermoid cysts. It is therefore likely that the prognosis for spinal epidermoid cysts is dictated by the extent of surgical removal and location relative to the spinal cord. Finally, recurrence of clinical signs can be expected following subtotal resection and regular follow-up MRI scans and examinations are recommended.The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author/s.Ethical review and approval was not required for the animal study because written informed consent was obtained from the owners for the participation of their animals in this study.DD contributed to the writing of the manuscript and literature review. MAM contributed to the histopathological examination, interpretation, and description of biopsy samples. MM contributed to the interpretation and preparation of MRI images. DD, ML, and ST contributed to the preparation of the manuscript. All authors reviewed and edited 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Rational antibiotic treatment of urinary tract infections (UTI) is important. To improve the quality of antibiotic treatment of UTI, it is essential to obtain insight into diagnostic approaches and prescribing patterns in general practice. The aim of this study was to investigate the quality of diagnostics and treatment of UTI in general practice by means of quality indicators (QIs). QIs provide a quantitative measure of quality and are defined by a numerator (the number of patients receiving a specific investigation or treatment) and a denominator . For adult patients with suspected UTI, practices registered the following: age, sex, risk factors, symptoms and signs, examinations, diagnosis and treatment. The levels of the QIs were compared with their corresponding standards. Half of the patients diagnosed with lower UTI or pyelonephritis fulfilled the diagnostic criteria for UTI: characteristic UTI symptoms and clear signs of bacteriuria, respectively. Urinalysis was performed for nearly all patients, including patients without characteristic symptoms of UTI. One-fourth of the patients with suspected lower UTI were treated with antibiotics despite no urinalysis and nearly half received antibiotics despite an inconclusive dipstick test. Pivmecillam was the preferred antibiotic. The findings of this study indicate that there is room for improvement in the management of UTI in Danish general practice. Antibiotic resistance has a major impact on human health worldwide and is mainly driven by the use of antibiotics . About 8The prevalence of urinary tract infections (UTI) varies depending on different risk factors such as age and gender. In Denmark, between 5% and 8% of women aged 40\u201345 years have UTI on a given day ,7,8. TheA total of 101 out of the 970 invited practices participated in the study (10.4% response rate). A total of 3076 consultations with patients suspected of having UTI were registered.At the index consultation, 1514 patients were labelled as \u201cunresolved\u201d . A total of 1263 patients were diagnosed with lower UTI and 16 patients with upper UTI (pyelonephritis). The remaining 219 patients were labelled as \u201cnot UTI\u201d, and 64 had a missing diagnosis.Two out of the eleven indicators were within the standard. Nearly all the patients suspected of having UTI who did not have any UTI symptoms  nor any systemic signs of infection , nevertheless, had a urinalysis performed  (QI1). Almost 90% of patients diagnosed with lower UTI had at least one lower urinary tract symptom (QI3). Nonetheless, only about half of the patients diagnosed with either lower UTI or pyelonephritis had UTI symptoms and clear signs of bacteriuria (dipstick positive nitrite and leucocytes and/or positive microscopy) (QI2 and QI5). Most patients diagnosed with complicated lower UTI or pyelonephritis had a urine culture and susceptibility test performed (QI4 and QI6).One out of the six QIs was within the standard. A total of 26% of patients with suspected lower UTI were treated with antibiotics despite the lack of a urinalysis (QI12). Among patients with UTI symptom(s), 11% of those with a negative urinary dipstick  and nearly half of those with an inconclusive urinary dipstick  were treated with antibiotics (QI13 and QI14).Four out of six QIs were within the standard. A total of 83% of patients without a penicillin allergy treated with antibiotics were prescribed pivmecillinam (QI19). Pivmecillinam was also the most frequently prescribed antibiotic for pregnant women and patients diagnosed with pyelonephritis (QI21 and QI24). Very few patients were treated with ciprofloxacin (QI20 and QI23).According to the diagnostic process, nearly all patients had a urinalysis performed at the index consultation. However, inappropriately, this also included most patients presenting to general practice with no UTI symptoms (QI1). Only about half of the patients who were diagnosed with lower UTI or pyelonephritis fulfilled the two diagnostic criteria: having the characteristic UTI symptoms and a urinalysis indicating bacteriuria, respectively (QI2 and QI5). Regarding the treatment decision, one-fourth of patients with suspected lower UTI were treated with antibiotics despite the lack of a urinalysis (QI12). Nearly half of the patients with lower urinary tract symptom(s) were treated with antibiotics even though they had an inconclusive urinary dipstick  (QI14). The majority of patients treated with antibiotics were prescribed pivmecillinam, and very few were prescribed ciprofloxacin (QI19 and QI20).To our knowledge, this is the first study to investigate the quality of the diagnostic process and antibiotic treatment of UTI in general practice by means of validated Qis. Another important strength is the large number of patients and practices included in the study. The data collection process was simple and fast, thus securing the continuous inclusion of patients.The healthcare professionals who volunteered to register their consultations may have been particularly interested in the management of patients with UTI and may have been more likely to follow guidelines compared to non-participants. Hence, quality may look better in the present data than in Danish general practice in general, which merely underlines the deviations from quality standards found in the present study.A total of 23 out of the 24 validated QIs were applicable to the data collected using the registration chart. As information about changing the catheter before the start of antibiotic treatment was not available in the APO registration chart, QI17 could not be applied.The construction of two of the indicators might not sufficiently reflect the widespread use of microscopy in Danish general practice (QI13 and QI14). Microscopic verification of uropathogenic bacteria in a patient with urinary tract symptoms may have led to antibiotic prescribing in spite of a negative dipstick. However, when removing patients with a positive microscopy from the two indicators above (QI13 and QI14), we still found a substantial number of patients treated with antibiotics in spite of a negative or inconclusive dipstick.Only data from the index consultation were collected. Consequently, we were not able to investigate the quality of any antibiotic treatment prescribed after receiving the results of urine culture and/or susceptibility testing. However, most antibiotics are prescribed on day one . Thus, iA total of 476 patients 15.5%) included in the study had none of the genitourinary symptoms attributable to UTI, nor any systemic signs of infection. Nevertheless, nearly all of these patients had a urinalysis performed with either a dipstick, microscopy and/or urine culture (QI1). Similar results were obtained in another recent Danish study, which found that the majority of patients without UTI symptoms had a urine culture performed . Accordi.5% incluUTI are defined by the presence of UTI symptoms in combination with a significant amount of uropathogenic bacteria in the urine .We found that only half of the patients fulfilled the two diagnostic criteria for having UTI: the presence of UTI symptoms and clear signs of bacteriuria (dipstick showing positive nitrite and positive leucocytes and/or positive microscopy) when diagnosed with lower UTI (QI2). Similar results for patients diagnosed with pyelonephritis were found (QI5), indicating a certain degree of mislabelling of both upper and lower UTI.Time pressure, consultation over the phone, and patient treatment expectations are possible explanations for antibiotic prescribing without the result of a preceding urinalysis. These factors are identified as some of the barriers to appropriate antibiotic prescribing . Only abWe found that 11% of patients with lower urinary tract symptoms were treated with antibiotics despite having a negative dipstick (leucocyte and nitrite) (QI13). The probability of having UTI for these patients is low ,27.A total of 42% of the patients with the symptom(s) of lower UTI who had an inconclusive urinary dipstick (positive leucocytes and negative nitrite) were treated with antibiotics QI14). Little et al. found a 75% probability of having UTI when only leucocytes are positive on the dipstick . Holm su. Little Nearly all patients diagnosed with pyelonephritis were treated with antibiotics  (QI15). This indicates that when GPs suspect \u201cpyelonephritis\u201d they prescribe antibiotics. However, only half of the patients diagnosed with pyelonephritis presented typical symptoms of pyelonephritis and bacteriuria (QI5). This indicates mislabelling leading to the overprescribing of antibiotics. Nevertheless, the underprescribing of antibiotics for pyelonephritis may also take place. Less than half of catheter users with the symptom(s) of pyelonephritis were treated with antibiotics (QI16). In line with our findings, Sommer-Larsen et al. showed that in a nursing home, only about one-third of patients with symptoms indicative of pyelonephritis received antibiotics .A total of 83% of patients with suspected lower UTI and no penicillin allergy treated with antibiotics were treated with a first-choice antibiotic (pivmecillinam) (QI19). In line with our findings, a study from 2019 describing the prescriptions for UTI in general practice in Denmark found that pivmecillinam was the most common antibiotic prescribed for lower UTI (45.8% of the prescriptions) . InteresWe found a very small proportion (1%) of quinolones prescribed for lower UTI (QI20). In comparison with other countries, higher quinolone prescription rates are seen: 22% in Belgium, 7% in the Netherlands, 3% in Sweden and 37% in Switzerland ,30.This study is part of a larger quality improvement project originating from Audit Project Odense (APO) with the overall aim of improving the quality of the diagnosis and treatment of UTI in general practice ,32. All The registrations were performed on A4-size paper registration charts. For each patient, information about the patient\u2019s age, sex, risk factors, symptoms and signs, examinations carried out, diagnosis given and actions made  were recorded in the registration chart .A set of 23 out of 24 validated QIs for the management of patients aged \u226518 years with suspected UTI was applied to the APO dataset . An indiData from the registration chart were used to calculate the levels of the QIs. The proportion of relevant observations fulfilling each of the 23 QIs was calculated as a percentage and compared with its corresponding standard. A 95% confidence interval (95%CI) for the corresponding odds was estimated using the method of generalised estimating equations (GEE) to adjust for correlation within practices; these were transformed back with the logit formula to obtain the 95%CI for the percentages. The data were analysed using SAS 9.4.This study presents an analysis of the quality of care for patients suspected of having UTI in Danish general practice. Urinalyses, such as dipstick, microscopy, culture and susceptibility testing, were found to be used excessively. The findings also indicate antibiotic overuse. However, high adherence to guidelines was found regarding the choice of antibiotic prescribed. Importantly, antibiotic underprescribing was also implied in some cases.The results of the study are based on the application of a set of QIs. Though it is important to keep in mind that QIs are used to generate reflection and debate about the quality of care rather than a definitive judgment of quality.The QIs applied in this study represent a useful tool for evaluating and comparing the quality of the diagnosis and antibiotic treatment of patients presenting with suspected UTI.The findings of the study can be used as a basis for future interventions to improve the quality of management of patients with suspected UTI. Further research should focus on the application of the indicators on a larger scale as part of a system-wide quality investigation program accessible to all general practices in Denmark. The application of the indicators to general practices in, for example, the Nordic countries may also offer an important opportunity to improve care."}
+{"text": "Many maternal and neonatal deaths are largely preventable by expanding the continuum of care . Even though ensuring the Continuum of Care (CoC) has advantages over separate services, evidence from the globe suggests that completion of the CoC for maternal health is very low. From our search of the literature, there is limited evidence on the completion of the entire CoC and its associated factors in sub-Saharan Africa (sSA). Therefore, this study aimed to assess coverage and associated factors of completion of the CoC for maternal health in sSA.Data for the study were drawn from a recent nationally representative survey of 32 Demographic and Health Surveys (DHS). A total weighted sample of 225,135 women of reproductive-age, who gave birth in the two preceding years were included. Due to the hierarchical nature of DHS data, a multilevel logistic regression model was applied to investigate individual and community-level factors that may influence completion of CoC. Adjusted Odds Ratios (aORs) with 95% Confidence Interval (CI) were reported and variables with 95% CI not including 1 were considered as significant factors of the completion of CoC.Only, 56,172  of the women in sSA utilized the CoC for maternal health which varied from 11,908 (17.9.0%) in East Africa to 7,418 , aged\u2009\u2265\u200935\u00a0years , attending primary education , secondary education , higher education , having mass media exposure , women from female-headed households  and women from communities with high maternal education . However, perceiving distance from the health facility as a big problem , residing in rural areas , delayed ANC initiation  and unintended pregnancy  were associated with lower odds of CoC.This study showed a low proportion of women, who utilized the CoC in sSA. Both individual and community-level factors were associated with CoC completion rates among women in sSA. Therefore, policymakers in sSA must consider both individual and community-level factors and undertake multi-sectorial approaches to address barriers of CoC at different levels.The online version contains supplementary material available at 10.1186/s12884-022-04757-1. Although the Maternal Mortality Ratio (MMR) declined from 342 per 100,000 live births in 2000 to 211 deaths per 100,000 live births in 2017 globally, most sub-Saharan African (sSA) countries had difficulties in achieving the Millennium Development Goals (MDGs 4 and 5) . MoreoveMany strategies have been implemented in an attempt to reduce maternal and child deaths . ContinuCurrently, CoC for maternal and newborn health (MNH) care is recommended as advantageous over each service provided separately to achieve the global target of ending preventable maternal, newborn and child deaths because each stage of the CoC builds on the success of the previous stages , 14, 15.Even though complete exposure to CoC has advantages over separate stages of care \u201320, evidSeveral studies investigated associated factors of maternal health care utilization in sSA \u201336. MostAll 33 Demographic and Health Surveys (DHSs), conducted in sSA from 2010 to 2018, were used. Only one DHS from Mozambique was excluded because it had no observations regarding PNC. We appended these datasets together to investigate completion and factors of CoC among women in sSA. DHSs are nationally representative surveys, conducted at five years\u2019 intervals across low- and middle-income countries , 41. Eac. This study used pooled data of the DHS surveys from 32 sSA countries. A total weighted sample of 225,135 women of reproductive age who gave birth was included.A two-stage stratified cluster sampling procedure was used to select study participants. In the first stage, Enumerations Areas (EAs) were selected based on the sampling frame of each country. In the second stage, a sample of households is selected from each EAs. The detailed sampling procedure used by DHS has been documented elsewhere . DHS surCompletion of a CoC which was a composite score of ANC, SBA and PNC, was the dependent variable. It was dichotomized as complete if women had received at least four antenatal care visits (ANC4\u2009+), SBA and PNC and incomplete if the women did not receive at least ANC4\u2009+\u2009, SBA or PNC. Current (2016) WHO recommendations on ANC modified the minimum number of ANC contacts from four to eight. In this study, however, the frequency of ANC contacts was measured dichotomously as less than four ANC visits and at least four ANC visits since many DHS surveys were conducted before 2016 . SBA wasBased on previous literature \u201331, 46 aExtraction, recoding and analysis of data were performed using Stata version 14 software. We summarized data by text, tables and figures. Data were weighted to assure representativeness of the survey and to get reliable statistical estimates (robust standard error). Weighting was done using the complex sample design weighting  or \u201csvyset\u201d Stata command. We conducted a multilevel analysis since DHS data have been collected in nested units. For the purposes of the analysis, we fitted four models. First a null model without any explanatory variable was fitted to assess outcome variability between clusters. Then, model 1 was fitted with individual-level variables only and model 2 with community-level variables only. Finally, model 3 was fitted by incorporating both individual-level and community-level variables. Intra-class Correlation Coefficients (ICC), Median Odds Ratios (MOR), and the Proportional Change in Variance (PCV) were estimated to measure CoC variation across clusters. Model comparison was based on deviance (-2LLR) and the best-fit model was considered a model with the lowest deviance.To select variables for multivariable analysis we fitted unadjusted regression models  for each independent variable. Variables with p-values\u2009\u2264\u20090.2 were selected for multivariable analysis  had planned their recent pregnancy, 200,886 (89.2%) were currently in union and 122,301 (60.9%) initiated the first ANC visit after 12\u00a0weeks of gestation. Regarding educational status, 89,422 (39.7%) women had no education and 70,817 (31.5%) had completed primary level. Nearly two-thirds, 146,212 (65.0%) had exposure to at least one media during the week  and 150,447 (66.8%) were rural residents. The largest portion of the women who received antenatal care had their blood pressure measured  and received tetanus injection   women attended at least one ANC visit during pregnancy. It was the commonest care received by women in each country which varied from 62.6% in Chad to 99.2% in Rwanda. The proportion of women with\u2009\u2265\u20094 ANC visits decreased to 54.5%, ranging from 31.1% in Chad to 87.2% in Ghana. The proportion of women who received\u2009\u2265\u20094 ANC visits and SBA was 42.8% . The proportion of women who received SBA and PNC decreased to 31.4%   indicating the effects of community heterogeneity were high as compared with the final model model 3). In model 3, as indicated by the PCV, 50% of the variation in maternal health care along the CoC across communities was explained by both individual and community-level factors. Also, model fitness was compared using deviance and the model with the lowest deviance (model 3) was considered the best-fitting model  among women aged 25\u201334\u00a0years and 1.40  among\u2009\u2265\u200935\u00a0years as compared to women aged 15\u201324\u00a0years. The odds of completing CoC were 1.44  for mothers who had primary education, 1.95  for secondary education and 2.15  for higher education as compared to those without formal education. Moreover, the odds of completing CoC was higher among women who headed female households , were exposed to mass media  and with high community education .The odds of completing CoC among women who reside in rural areas were 22% lower  as compared with women who reside in urban areas. The odds of completing CoC among women reporting distance to a health facility as the big problem was decreased by 12%  compared with women who deem distance to a health facility not a big problem. Similarly, women who did not intended to be pregnant had lower odds of completing CoC as compared to those with planned pregnancies . In addition, women who started ANC after 12\u00a0weeks of gestation had lower odds of completing CoC as compared to those who initiated ANC before 12\u00a0weeks of gestation   of the women in sSA. This was higher in Southern Africa with 51.5%  and much lower with approximately 20% in the other three regions. Large disparities of CoC were found across sSA countries with as low as 2.7% in Burundi and as high as 61.2% in South Africa. This finding is in line with pooled estimates of South Asian countries (24.5%) . Our finThis study demonstrated that the inclusion of community-level variables was important in explaining the variations in the completion of CoC. Community-level variables such as region, residence, distance from health facilities and community education showed effects on the completion of maternal health care. Only 2.27% of the total variation remained unexplained after adjustments of individual and community level factors.When maternal age increases, the odds of completing CoC increases, consistent with some previous studies , 52. SomConsistent with studies in Ethiopia, Nigeria, Ghana, Chad, Gambia, Nepal, Pakistan, higher women\u2019s education was significantly associated with increased odds of CoC , 51, 52.Supported by studies conducted in Ethiopia, Chad, Pakistan and Nepal, women exposed to mass media were more likely to utilize CoC , 59, 60.As observed in previous studies, lower odds of completing CoC were observed among women in rural areas as compared to urban areas , 59, 66.Women with unintended pregnancies had lower odds of completing CoC as compared to planned pregnancies, similar to studies in Ethiopia, Ghana and Nepal , 29, 30.Delayed initiation of ANC was negatively and significantly associated with utilization of CoC, in agreement with studies from Ethiopia, Gambia, and Japan , 36, 48.The odds of completing CoC was found to be higher among women from communities with a high percentage of educated women. Low literacy levels in the community may be related to low health knowledge, while women from highly educated communities may acquire information from others regarding benefits of maternal health care, leading to increased odds of completing CoC. Illiterate women are economically unstable and may fail to receive adequate maternal health care during pregnancy .Strengths of this study are first of all large sample size from nationally representative data from 32 sSA countries with appropriate multilevel statistical analysis. Second, our study applied a more comprehensive measure of maternal health care that collectively considered access to CoC. Our findings, however, cannot provide information on causality as our study had a cross-sectional design. Measurement of the main components of CoC is self-reported based on women\u2019s recall and this may have led to recall and social desirability bias. Due to the secondary nature of data, we did not include some factors which may influence utilization along the CoC, such as quality and satisfaction with care, knowledge of maternal health care and danger signs. Besides, it was unable to assess complete PNC within six weeks after birth of women and newborns as an element of CoC because such data were not collected in DHSs.Low proportions of women utilizing CoC in sSA were observed with the highest coverage in Southern Africa and almost similar low coverage in East, West and Central Africa. Both individual and community-level factors were associated with CoC completion. Factors associated with high CoC completion rates, include older age, having attended education, having mass media exposure, intended pregnancy, timely ANC initiation, female-headed households,, perceiving distance from health facilities not as big problem and residing in urban areas and from highly educated communities. These findings point to areas where care can be better tailored to improve the completion of CoC. Policymakers in sSA must consider both individual and community-level factors and undertake multi-sectorial approaches to address barriers at different levels. Thus, those living in rural areas, less educated, initiated ANC lately, perceiving distance from health facilities as big problem and from communities with low education need more attention to increase completion of CoC and improve maternal and newborn health.Additional file 1.Additional file 2."}
+{"text": "The surgeon\u2019s experience did not show a significant difference through the Kruskal\u2013Wallis test in the femur  and the tibia . The stage of arthrosis only revealed a significant difference in the planning of the femur  alone. Discussion/Conclusion: Digital templating for total knee arthroplasty brought up gender differences, with oversized implants for women and undersized implants for men. A high stage of femoral arthrosis can lead to the under and oversized planning of the surgeon. Since the surgeon\u2019s experience in planning did not show an effect on the adherence to templating, the beneficial effect of digital templating before surgery should be discussed.Introduction: Preoperative digital templating is a standard procedure that should help the operating surgeon to perform an accurate intraoperative procedure. To date, a detailed view considering gender differences in templating total knee arthroplasty (TKA), stage of arthrosis, and the surgeons\u2019 experience altogether has not been conducted. Methods: A series of 521 patients who underwent bicondylar total knee arthroplasty was analyzed retrospectively for the planning adherence of digital templating in relation to sex, surgeon experience, and stage of arthrosis. Pre- and postoperative X-rays were comparably investigated for planned and implanted total knee arthroplasties. Digital templating was carried out through mediCAD version 6.5.06 . For statistical analyses, IBM SPSS version 28  was used. Results: The general planning adherence was 46.3% for the femur and 41.8% for the tibia. The Mann\u2013Whitney U test revealed a gender difference for templating the femur (z = \u22125.486;  Worldwide, total knee arthroplasty is a popular surgical intervention  as a pos\u00ae  in consecutive order. The data were collected from the digital patient documentation system, the digital patient archive system, and the digital radiography system and were recorded metrically, ordinally, and nominally.The present study was performed according to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) . This stPatients undergoing primary cemented bicondylar total knee arthroplasty were examined and their eligibility to participate in this study was assessed. The inclusion criteria were (1) patients with primary and secondary arthrosis undergoing a cemented primary knee arthroplasty, (2) cemented primary bicondylar total knee arthroplasty, (3) accessible patient data, (4) implantation of S&N Genesis II CR/PS, Journey II BCS/CR, and (5) TKA: anteromedial approach (with median skin incision). The exclusion criteria were (1) revision knee arthroplasty, (2) uncemented primary knee arthroplasty, (3) inaccessible patient data, and (4) a different approach than anteromedial. The present study was approved and registered by the ethics committee (HDZ-NRW) of the RUB University of Bochum and conducted according to the principles expressed in the Declaration of Helsinki. For an overview, see A standardized preoperative digital radiograph of the anterior-posterior view of the full leg in a standing position (with a view of the hip and ankle joint), a lateral view of the knee, and a patella defile view were taken of the patients. A standardized metallic radiopaque ball with a diameter of 25 mm was used as a reference for determining the magnification factor.\u00ae version 6.5.06  in a standardized manner. First, the full leg stand view was used for planning. Afterward, the lateral scan was used for planning purposes. Initially, scaling at 25 mm was performed automatically by the software with the metallic radiopaque ball as the reference. Then, the correct operation side was marked in the software which unlocks the button for the implant types and sizes. After choosing the correct implant type, the implant size was selected by the surgeon. For the full leg stand view, implants in an anterior-posterior view were planned and for the lateral view, implants in a mediolateral view were planned. For the femoral side, the position was set at a beta angle of 5, 6, or 7 degrees. For the tibia, the correct position in line with the ankle joint was important. In general, the digital template was adjusted to the size in the X-ray and should not exceed the size shown in the X-ray. Once the tibia and femur had been planned, the image was saved and transferred to the digital radiology system.Digital templating was carried out with mediCADThe age, sex, side, body mass index (BMI), length of hospital stay, time of operation, type of implants, size of the implants pre- and postoperative, the experience of surgeons (classified through the quotient of the number of surgeries from the particular surgeon divided by the number of surgeries from the surgeon with the highest amount of TKA), and grade of arthrosis (Kellgren\u2013Lawrence score) were recorded. Data analysis between sex, the experience of the surgeons, the Kellgren\u2013Lawrence score, and the difference between the planned and implemented size were performed.p-values < 0.05 show significance. Furthermore, linear regression with an effect strength of R2 (Cohen)  was used to control the results. Through regression analysis, it was investigated whether the degrees of experience or arthrosis have a linear influence on planning adherence.All statistical analyses were performed using the software IBM SPSS version 28 . The significance level was set two-sided with \u03b1 = 0.05. Age, BMI, length of hospitalization, length of intensive care stay, pre- and postoperative laboratory parameters, and frequency of transfusion were listed metrically. Sex, side, preconditions, and systemic and surgical complications were listed nominally. For patients, demographic data were analyzed by the mean, standard deviation, and percentage. For general planning adherence, the planned size of the femur and tibia was subtracted from the implanted size. The exact planning adherence was 0. For planning adherence and sex, experience, and the degree of arthrosis, the nominal, dichotomous data were analyzed by Fischer\u2019s exact test. This test was used because of its independence from the sample size. The additional effect size  was used to detect the clinical impact of a significant result. For planning adherence and sex, each number from femur +III to \u2212II was analyzed with sex. In addition, the exact match, above, and under sizes from the femur and tibia were listed ordinally and analyzed through the Mann\u2013Whitney U test to detect differences between sizes and sex and support Fischer\u2019s exact test. The z-values of the Mann\u2013Whitney U test reflect the standard deviations. A z-value > \u22122 or 2 indicates that measured significances cannot be explained by theoretical random patterns. For planning, adherence, experience, and degree of arthrosis, each experience level or degree of arthrosis was analyzed with Fischer\u2019s exact test. To support the results, the Kruskal\u2013Wallis test was used because it enables the evaluation of more than three independent samples. The asymptomatic p and Kruskal\u2013Wallis H values were used with regard to the significance level. Kruskal\u2013Wallis H values above 5.99 and asymptomatic n = 865 patients with primary knee arthroplasties from the years 2018\u20132021 during the inpatient stay and follow-up were analyzed. In total, N = 344 patients with cemented and uncemented primary unicondylar knee arthroplasty and primary cemented bicondylar arthroplasty with primary cemented implants other than Journey II or Genesis II were excluded. An in-depth analysis of n = 521 was performed. For an overview, see For this retrospective study, data from n = 521 patients were analyzed. Overall, 36.1% (188 of 521) of these patients were men and 63.9% (333 of 521) were women. The mean age was 70.37 (\u00b19.2). In 96.4% (n = 502) of the cases, primary arthrosis was the indication, and in 2.7% of the cases, secondary arthrosis was the indication. A total of 17.5% (n = 91) showed a severe varus in the arthrosis, and 13.1% (n = 68) had a severe valgus in the arthrosis. No patient showed a type I Kellgren\u2013Lawrence score. However, 2.3% (n = 12) were classified with a type II Kellgren\u2013Lawrence score, 29.6% (n = 154) with a type III Kellgren\u2013Lawrence score, and 62.4% (n = 325) with a type IV Kellgren\u2013Lawrence score. According to the WHO criteria, the mean BMI was 30.95 (\u00b16.71); 0.6% (n = 3) were underweight (BMI < 18.5 kg/m2), 13.8% (n = 72) were normal-weight (BMI \u2265 18.5 and \u226425 kg/m2), 35.5% (n = 185) were overweight (BMI \u2265 25 and \u226429.9), 23.0% (n = 120) were obese type I (BMI \u2265 30 and \u226434.9 kg/m2), 13.8% (n = 72) were obese type II (BMI \u2265 35 and \u226439.9 kg/m2), and 10% (n = 52) were obese type III (BMI \u2265 40 kg/m2). The mean pain symptom value before surgery was 6.4 points (\u00b12.21) (n = 259) and a day before leaving the hospital after surgery was 1.57 points (\u00b10.55) (n = 269). The mean ROM before surgery was 99.97 degrees (\u00b116.26) (n = 509), after surgery was 88.71 degrees (\u00b16.41) (n = 483), and at the time of re-appointment was 105.12 degrees (\u00b118.32). The mean length of stay was 10.13 days (\u00b13.64), and the mean operation time was 99 min (\u00b10.49 min). In total, 16 surgeons were listed for total knee arthroplasty from 2018 to 2021 and 34.9% Journey II implants (n = 182) and 64.5% Genesis II implants (n = 336) were implanted.Data from n = 241) of the cases. In 22.8% of the cases (n = 119) one size bigger, in 2.5% (n = 13) two sizes bigger, and in 0.4% (n = 2) three sizes bigger were planned. In 23.4% of the cases (n = 122), one size smaller, and 2.5% of the cases (n = 13) two sizes smaller were planned.Femur: 11 of 521 cases were missing. The exact planning for the femoral side was achieved in 46.3% (n = 218). One size bigger was planned in 9.6% (n = 50) and two sizes bigger were planned in 1.7% (n = 9) of the cases. One size smaller was planned in 20.3% (n = 106) of the cases, and two sizes smaller were planned in 1.9% (n = 10). For an overview, see Tibia: 126 of 521 cases were missing. The exact planning adherence of the tibia was fulfilled in 41.8% of the cases (n = 82) of the cases in male patients and 48.6% (n = 159) of the cases in female patients. Femoral plus I was implanted in 12.5% (n = 23) of the cases in male patients and 41.5% (n = 96) in female patients. Femoral plus II was used in 2.1% (n = 4) of the male cases and 2.8% (n = 9) of the female cases. Femoral plus III was implanted in 0.5% (n = 1) of the male cases and 0.3% (n = 1) of the female cases. Femur minus I was used in 37.1% (n = 68) of the male patients and 16.5% (n = 54) of the female cases. Femur minus II was found in 2.7% (n = 5) of the male cases and 2.44% (n = 8) of the female cases. Overall, there was a significant difference in implanted sizes in relation to sex . For significant group differences in detail, see Femur: The exact adherence of the femur implant was achieved in 44.8% (n = 63) of the male cases and 60.5% of the female cases. Tibia minus I was implanted in 37.4% (n = 52) of the male cases and 21.0% (n = 54) of the female cases. Tibia minus II was used in 3.5% (n = 5) of the male cases and 1.9% (n = 5) of the female cases. Tibia plus I was found in 11.5% (n = 16) of the male cases and 13.2% (n = 34) of the female cases. Tibia plus II was implanted in 1.4% (n = 2) of the male patients and 2.7% (n = 7) of the female patients. Altogether, there was a significant difference in the implanted sizes in relation to sex Tibia: The exact adherence of the tibia implant was achieved in 45.3%  and the tibial side showed no significant relation to the planner\u2019s experience . In addition, the linear regression did not show a significant relation adherence to the experience of the surgeon . Group 2 (<50%) revealed two significant results for femoral plus I and tibia minus II implantations against all other groups , and group I showed a significant result in less tibial minus II implantations against all other groups . Detailed group differences were calculated through the exact Fisher\u2019s test. For more details, see The experience of the surgeons was classified through the number of surgeries. The surgeon with the most operations was set as the reference (100%). All procedures of the reference surgeon were divided by the surgeries performed by the other surgeons. Four categories were formed: (4) > 75%, (3) < 75%, (2) < 50%, and (1) < 25%. Overall, there was no surgeon categorized in group 3. Regarding experience, the femoral side was not significantly different  were classified as II, 29.6% (n = 154) were classified as III, and 62.4% (n = 325) were classified as IV. In general, the femoral side showed a significant difference between the planning adherence and the K\u2013L score , whereas the tibial side showed no significant difference . The K\u2013L-score III was significantly more related to femur plus I implantation  against all other groups. Linear regression could not show a connection between all implantation matches to the degree of arthrosis . For the femoral side, n = 37 cases were missing. For the tibial side, n = 151 cases were missing  and computed tomograph (CT) planning methods are also shaping the market ,14,15. DFor 2D-templating, Klag et al., 2022 revealed only a 50% prediction rate for surgeons for the tibial side and a 77% prediction rate for the femoral side ,18. In tThe influence of the experience of a surgeon in digital templating can be a decisive factor in the success of arthroplasty ,20. The Digital templating and sex are discussed in the literature. Other studies could not reveal any gender differences in relation to planning ,21. Sincp = 0.038). Additionally, especially for extremely deformed joints, a precisely planned implant could be helpful for the surgery and might reduce the time of the operation. As a result, in the future, more 3D templating will be established. Accuracies of up to 90% and independence from different patient weights against 2D planning have been demonstrated already [As the degree of arthrosis for a total arthroplasty is mostly in the final stage, a comparative analysis in the literature was barely found . Regardl already ,28.However, this study has its limitations. First, this single-center analysis could only image the planning strategy of the in-house surgeons. Second, from the 521 analyses, some planning details for the femur and tibia were missing, which lead to a reduction in the patient count in the results. Third, there is a difference in the count of men and women, which could be a bias factor. Fourth, the planner\u2019s experience was categorized through a hospital\u2019 s internal calculation; other hospitals could use a different categorization method. Fifth, the classification of the stage of arthrosis is dependent on the subjective opinion of the indicating surgeon and could thus lead to a distortion of the results.The 2D digital planning of total knee arthroplasty shows a gender difference in the exact templating. The more frequent undersized implants for males and oversized implants for females should be noted. The surgeon\u2019s experience does not influence the exact templating of implants. Planning the femoral component in high-stage arthrosis is more related to under and oversizing. As the general planning adherence was under 50%, it should be questioned if conventional digital planning has a deep impact on the total knee arthroplasty. There is a further need for planning tools that could help surgeons to plan more accurately. Currently, low-dose CT 3D scans, MRI 3D scans, or 3D reconstructions from 2D X-rays are in use to improve digital templating ,30,31. A"}
+{"text": "Exercise and cold exposure are two stimuli that have been suggested as solely effective to modulate adipose tissue metabolism and improve metabolic health in obese populations. The two primary organs involved in energy metabolism during exercise and/or cold exposure are skeletal muscle and adipose tissue. Adipose tissue can be divided mainly into two types: white adipose tissue (WAT), which primarily stores energy, and brown adipose tissue (BAT), known as the primary source of thermogenesis. The exercise-stimulated release of myokines allows for crosstalk between skeletal muscle and adipose tissue, partially mediating the beneficial effects of exercise. Cold exposure is another trigger for the regulation of myokine secretions, thus increasing adipose tissue metabolism, especially via activation of BAT. Therefore, this has generated the hypothesis that exercise in conjunction with cold exposure might be the optimal regimen to regulate myokine profiles and gain more beneficial health effects. However, to date, human experimental data regarding different exercise  and cold exposure  patterns are scarce. In this review, we will summarize the current human clinical trials investigating the regulation of myokines induced by exercise combined with cold exposure, to elaborate on the roles of myokines in mediating adipose tissue metabolism. Although obesity has been studied for several decades, the overall prevalence has still increased dramatically and has affected a large number of people all over the world. Obesity is a strong risk factor for several chronic diseases, including type 2 diabetes, insulin resistance, hypertension, sarcopenia and some types of cancer . It has Most exercise intervention studies were carried out in a neutral thermal environment 20\u201325 \u00b0C), but temperatures higher or lower than neutral temperature can have an impact on exercise performance and energy metabolism. Due to global climate change, negative health effects caused by hot ambient conditions have been widely studied [0\u201325 \u00b0C, A few studies have demonstrated the powerful capability of cold exposure or cold acclimation to improve the metabolic profile. For example, daily cold exposure increased the brown adipose tissue (BAT) volume (mL) and oxidative capacity in humans . In addiBoth exercise and cold exposure can induce the secretion of some circulating factors, which play roles in altering metabolic homeostasis and insulin resistance ,8. So, tInterestingly, cold exposure has also been reported to facilitate modulating the expression and release of myokines, thus increasing adipose tissue metabolism . In thisSimilarities in the health benefits of exercise and cold exposure are mainly related to adipose tissue metabolism. Mammalian adipose tissue can be divided into two types: white adipose tissue (WAT) and BAT. WAT primarily stores energy as triglycerides, while BAT is known to dissipate energy by upregulation of the expression of mitochondrial uncoupling protein 1 (UCP1), a BAT-specific activation marker . UCP1 unIn addition to its effects on skeletal muscle and the cardiovascular system, exercise was recently demonstrated to result in adaptations of adipose tissue, and, therefore, improve whole-body metabolic health. Conflicting results exist regarding the effects of exercise on BAT between human and animal studies . HoweverAdditionally, cold exposure was reported to increase beta-adrenergic and/or UCP1 activity, thereby enhancing thermogenesis and fat metabolism in BAT . The facOther than browning, both exercise and cold are involved in the regulation of lipid metabolism. Although the effects of exercise on BAT metabolism have not been studied thoroughly, WAT lipolysis increases significantly after exercise in both rodents and humans ,23. CyclThe therapeutic potential of myokines in metabolic diseases is evident. By linking skeletal muscle to other organs , myokines can explain positive alterations in response to stimuli. The expression and release of myokines are affected by both exercise and environmental temperature, thus mediating adipose tissue metabolism. An overview of the exercise- and cold-regulated myokines to be discussed is given in Irisin, a contraction-regulated myokine, was first identified in 2012 as the secreted form of fibronectin type III domain containing 5 (FNDC5) mediated by the transcriptional coactivator PGC-1\u03b1 . As a noAlthough conflicting results still exist, both animal and human studies have shown that circulating levels of irisin were affected by exercise and low temperature ,27,38. Cmax) at 0 \u00b0C resulted in a more significant serum irisin increase [p = 0.06), upregulation of serum irisin was also observed after the Yukon Arctic Ultra, the longest and coldest ultra in the world, at \u221225\u2013\u22122 \u00b0C [max, 4 days/week) and 60 min of cycling at 15\u201319 \u00b0C (60% HRmax) and at 7 \u00b0C (60% Wmax) all showed no change in plasma irisin concentration [max (3 times/week) [Taken together, it is possible to increase irisin secretion and optimize the exercise effects by combining exercise and cold exposure. A study conducted by Ulupinar et al. in 2021 showed that aerobic running exercise   and 50 mes/week)  did not es/week) .Furthermore, it was reported that exercise-induced irisin secretion seemed to be accentuated in older adults and increased in response to cold exposure in obese subjects . HoweverFGF21, released mainly by hepatocytes , can alsThe FGF21 response to exercise is still ambiguous. Despite inconsistent results following exercise ,49,50, cmax (15\u201319 \u00b0C) and the Yukon Arctic Ultra (\u221225\u2013\u22122 \u00b0C), respectively [max) caused a significant increase in serum FGF21 concentration, whereas in combination with WBC , it abolished this effect [Considering exercise in cold environments, plasma and serum FGF21 levels were unchanged after 60 min of cycling at 60% HRectively ,43. Compectively . Therefos effect  (Table 2max), a reduction in glucose levels was shown [The main tissue source of FGF21 after exercise is still unknown. It was suggested that exercise is a key stimulus in inducing the hepatic, but not skeletal muscle, release of FGF21 into the systemic circulation . Differeas shown . The eviAs the classic and best-characterized myokine, significant amounts of IL-6 were proved to be released from contracting skeletal muscle during prolonged exercise . ExercisAlthough, plasma concentrations of IL-6, which did not increase with cold exposure, significantly lower WAT UCP1 protein content in IL-6 KO mice, indicating the important role of IL-6 in regulating cold-induced UCP1 expression ,58, thusAs a blood inflammatory marker, IL-6 was mainly investigated with cryotherapy in combination with exercise. Ziemann et al. reported that IL-6 levels increased after a 5-day WBC  in combination with a moderate-intensity training program for professional tennis players . HoweverAs an exercise- and cold-induced circulating factor in skeletal muscle and adipose tissue, respectively, Metrnl was first identified in 2014 . Unlike In rodents, Metrnl expression was affected by exercise type and muscle position. A 2-fold increase in circulating Metrnl levels was found post-eccentric exercise. However, in the same study, an endurance exercise training program did not change the Metrnl level . On the When combining exercise with cold exposure, the Metrnl level remained unchanged after the Yukon Arctic Ultra at \u221225\u2013\u22122 \u00b0C and even declined in overweight women after interval training for 40 min at 16.5\u201317.5 \u00b0C ,59 Tabl. The difMyostatin was the first identified myokine in 1997, which has been well-known for inhibiting skeletal muscle cell growth and differentiation . MyostatBoth endurance and resistance training lead to a decrease in myostatin levels . In addiLimited studies have investigated changes in myostatin in response to exercise coupled with cold exposure. Jaworska et al. first reported that WBC  following a 4-week resistance training program is effective in lowering circulating levels of myostatin . HoweverApart from the myokines mentioned above, some other circulating factors might be related to exercise and low temperatures, such as vascular endothelial growth factor A (VEGFA), brain-derived neurotrophic factor (BDNF), follistatin-like protein-1 (FSTL1) and lactate. These factors might also be potential therapeutic targets for metabolic diseases . HoweverAs an angiogenic factor, vascular endothelial growth factor A (VEGFA) is a major regulator of vascular endothelial cell activation, proliferation and migration. Various studies have shown that physical exercise upregulates VEGFA in mice and humans ,71,72. WAlthough mainly released from the brain, known as a factor improving cognitive function, BDNF was also identified as a contraction-regulated myokine capable of enhancing AMPK activation and, hence, lipid oxidation . It has As the well-known end product of anaerobic glycolysis, lactate levels can rise 20-fold during intense exercise. In addition to being an energy substrate, lactate also plays a role as a signaling molecule, delivering oxidative and gluconeogenic substrates ,81. Inte2max), as well as acute sprint interval training with high intensity [+/\u2212 mice and 3T3-L1 cells but not in young and healthy humans [Follistatin-like protein-1 (FSTL1) is an extracellular glycoprotein from the follistatin family, secreted by adipose, lung, heart and also primary human skeletal muscle cells ,83. It wntensity ,86. Furty humans ,87. The According to recent findings, it seems that environmental temperature during exercise can be used\u2009to increase adipose tissue metabolism by regulating myokine profiles. Elements of both exercise and environmental temperature need to be considered to develop an optimal exercise regimen.As a skeletal muscle contraction-induced myokine from skeletal muscle, irisin expression is upregulated when ST occurs in the cold. Key effects exist in exercise intensity and duration as well as temperature and cold exposure time. Unlike irisin, FGF21 is related to NST responses when exposed to prolonged cold. Exercise intensity also affects its expression. Mechanisms underlying the effect of cold exposure on IL-6, Metrnl and myostatin have not yet been investigated but animal models have demonstrated the possibility of increasing energy expenditure via regulating these myokines in the cold. Considering the contradictory results, data from animals should be transferred cautiously to humans. To date, there is not sufficient research on myokine responses to cold exposure, especially in humans. Future research needs to be conducted to clarify the mechanisms behind WAT browning induced by myokines. In addition, the effects of gender, age and human body composition (adiposity) on myokine concentration with cold exposure solely or combined with exercise warrant future investigation. Blood collection time, measuring method and intervention duration should be considered rigorously to improve the research quality. Further, although there are potent similarities between exercise and cold exposure, dissimilarities exist. Cold exposure triggers mechanisms in the human body to compensate for heat loss, while exercise increases heat production. Therefore, when the two stimuli are combined, the physiological responses become more complex. Whether antagonism exists between the two stimuli and which organ plays the main role in releasing the secretory factors remain to be investigated. To solidify the findings in humans, well-designed and controlled clinical studies are needed."}
+{"text": "The current diagnostic criteria for multiple sclerosis (MS) lack specificity, and this may lead to misdiagnosis, which remains an issue in present-day clinical practice. In addition, conventional biomarkers only moderately correlate with MS disease progression. Recently, some MS lesional imaging biomarkers such as cortical lesions (CL), the central vein sign (CVS), and paramagnetic rim lesions (PRL), visible in specialized magnetic resonance imaging (MRI) sequences, have shown higher specificity in differential diagnosis. Moreover, studies have shown that CL and PRL are potential prognostic biomarkers, the former correlating with cognitive impairments and the latter with early disability progression. As machine learning-based methods have achieved extraordinary performance in the assessment of conventional imaging biomarkers, such as white matter lesion segmentation, several automated or semi-automated methods have been proposed as well for CL, PRL, and CVS. In the present review, we first introduce these MS biomarkers and their imaging methods. Subsequently, we describe the corresponding machine learning-based methods that were proposed to tackle these clinical questions, putting them into context with respect to the challenges they are facing, including non-standardized MRI protocols, limited datasets, and moderate inter-rater variability. We conclude by presenting the current limitations that prevent their broader deployment and suggesting future research directions. Its halAs the manual detection of WML is time-consuming and prone to inter-rater variability , a myriaOne major drawback of the current MS diagnostic criteria is their lack of specificity, as they were proposed to identify patients with a high likelihood of MS rather than distinguish MS from other conditions . The lacQuantitative MRI, such as relaxometry, myelin imaging, or diffusion MR, provides information related to the microstructural composition and organization of tissues. In MS, quantitative MRI techniques complement conventional MRI techniques by providing insights into disease mechanisms . For insRecently, advances in MR technology, such as the development of specialized sequences, acceleration of protocols, and the proliferation of ultra-high field MRI, have allowed the imaging of pathologically specific MS lesional biomarkers . These iIn this review, we first briefly describe these advanced imaging biomarkers and their imaging requirements and then focus on image processing techniques tailored for their automated segmentation and classification. We conclude with a discussion on current limitations and future lines of research to boost the development of ML approaches in this area and encourage their adoption in MS research and clinical settings.2In this section, we present a brief description of CL, CVS, and PRL, and their respective imaging protocols. In addition to the CVS and PRL, which have emerged as promising MS biomarkers in recent years, we also included CL which, although included in the MS diagnostic criteria, are not yet commonly analyzed in clinical practice. For the sake of completeness, a short description of slowly expanding lesions (SELs) is also provided, although these have not been assessed with ML-based approaches yet.Cortical lesions (CL) - Cortical lesions are a type of MS lesions that involve, at least partially, the cortex and have been classified into three main categories  - Recently, studies have suggested that an MRI-detectable central vein inside MS lesions might be evidence of pathological processes specific to MS  - Recent pathology studies have demonstrated that about 30% of chronic demyelinated lesions are pathologically characterized by perilesional accumulation of iron-laden microglia and macrophages, showing evidence of smouldering demyelination and axonal loss around an inactive hypocellular core  \u2013 A different computational approach, designed to detect in vivo longitudinal volumetric lesional changes not associated with gadolinium enhancement, identifies the so-called \u201cslowly evolving/expanding lesions\u201d or SELs. Linear and radial lesion expansion is computed as a function of the Jacobian determinant of the non-linear deformation field between baseline and follow up scans (linearity assessment requires a minimum of 3 scans) (3 scans) . Advanta3 scans) . A recen3 scans) . The neu3 scans) .Overall, CL, PRL, and CVS have the potential to considerably improve the specificity of MS diagnosis . Moreove2.1Compared to conventional imaging biomarkers, the visual assessment of CL, PRL and CVS present some additional challenges.Imaging and assessment guidelines- The first obstacle is represented by the lack of consensus guidelines for imaging protocols. Although efforts have been made to standardize the use of MRI in clinical practice for conventional biomarkers , QSM, and multi-echo T2* GRE at both 3\u00a0T and 7\u00a0T  proposed a standard radiological definition and suggested specific MRI acquisitions . Followiisitions . Neverthisitions , there a and 7\u00a0T . HoweverThese evolving or unclear criteria for CL, the CVS, and PRL, wide variety of imaging settings, and lack of clear guidelines for standardized protocols clearly jeopardize the development and wide use of these biomarkers and of targeted ML techniques.Expert assessment - Even for experts, the task of segmenting CL, detecting the CVS, or classifying PRL is intrinsically more challenging than segmenting WML. CL are generally smaller in size and more affected by partial volume (PV) effects, compared to WML. The cortex is convoluted, so lesion shape is not as regular as in WM, and traditional methods of radiological evaluation (scrolling through an image stack) are less effective in this context. The detection of the CVS requires susceptibility-based MRI and its exclusion criteria need to be carefully considered when performing its assessment  . MSLAST EPI. A cohort of 60 patients was analyzed with a total of over 2000CL manually segmented by two experts. The CNN architecture proposed was similar to the one just described, but with a modified output. In addition to the CL segmentation, the CNN provided a classification into two types  and a separate branch with a simple tissue segmentation in WM/GM. CL were correctly classified into the two types by the network with an accuracy of 86%. Setting a minimum lesion size of 0.75\u00a0\u03bcL, it achieved a CL detection rate of 67% with, however, a quite high false positive rate of 42%  or CVS- (MS lesions without the CVS) have been proposed in the literature . Both apanalysis . Perivenanalysis , and a pMaggi, Fartaria et al.  introduc3.3EPI and phase 3D-EPI images. RimNet\u2019s architecture is inspired by the VGGnet (EPI image and one for the phase 3D-EPI image), where each CNN is made of three convolutional layers followed by a max-pooling operation. 3D patches of size 28x28x28 (centered around each MS lesion) are fed to each branch, and both high-level and low-level feature maps are concatenated. An automated lesion segmentation based on FLAIR and MPRAGE/MP2RAGE . The automated pipeline, after some pre-processing steps that included lesion segmentation  when considering a 50% CVS\u00a0+\u00a0lesions criteria to distinguish MS from MS mimics . A lower4.1Some common trends can be observed in most of the proposed pipelines. The large majority of the methods are supervised, relying on expert annotations. Regarding the DL-based approaches, they all used patch-based 3D CNN, exploiting the 3D intrinsic information, and often considered more than one MRI contrast simultaneously. In addition, a shared tendency consists of the use of relatively shallow architectures, with a limited number of trainable parameters, due to the lack of large datasets . CombiniIn addition, some common pre-processing steps can also be identified. First, some methods use intensity normalization techniques, either based on entire 3D volumes  or on si4.2Currently, a major limitation hinders the deployment of the above-described methods to the clinic: the methods proposed were trained and evaluated on small datasets acquired from one or at most two centers. Moreover, the MRI protocols used were often similar and not representative of the current diversity of images acquired in the clinics, including different processing, scans affected by noise and artifacts or protocols missing certain modalities. Therefore, the automated ML methods\u2019 robustness on larger datasets and different scanners, especially from multiple vendors, remains to be proven. This limitation is emphasized by the current lack of standardized acquisition protocols which increases the diversity of the MRI sequences considered for the same biomarkers. This also represents a major hurdle for potential regulatory approval of such methods. As regulatory approval is necessary for widespread adoption in the clinics, which is, in turn, the prerequisite for the availability of large datasets, this is currently a circular dependency issue.In addition, the achieved performance levels of the automated ML methods are still inferior compared to the human experts. Considering the high inter-rater variability and the limited amount of data available, there is also a considerable risk of having methods that perform well on data annotated by a single expert and not as well with annotations from other raters. To mitigate this issue, several methods have already considered consensus annotations from two or more experts . RegardiAnother main limitation is represented by the fact that some methods presented are not fully automated. CVSnet , for insFinally, for every automated tool the regulatory environment remains a critical barrier, as up to date less than 90 AI/ML-based medical devices or algorithms have been approved by the US Food & Drugs Administration (FDA). This challenge, however, is not unique to the three biomarkers considered  but shar4.3Standardization of the biomarkers\u2019 assessment- The first two necessary steps toward the improvement of the above-referred approaches are the validation of the biomarkers\u2019 specific criteria and standardization of the relative MRI protocols. CL have been recently included in the MS diagnostic criteria  and being evaluated on private datasets. In the future, the generalization of the proposed methods should be validated on large, multi-site datasets with standardized metrics. For this purpose, we urge research groups to organize grand challenges and release publicly available datasets with manual annotations of CL, PRL, and CVS. As already proved for several other tasks in medical imaging  methods are needed as to on one side provide uncertainty estimates regarding the output provided and on the other side transparency on the decisions taken by the DL-models. By explainability, we refer to a set of domain features such as pixels of an image or human-understandable high-level attributes that contribute to the output decision of the model and its internal working. To our knowledge, there are only two groups that have investigated XAI in MS. Eitel et al. , as the employer of C.G., has received the following fees which were used exclusively for research support: (i) advisory board and consultancy fees from Actelion, Genzyme-Sanofi, Novartis, GeNeuro and Roche; (ii) speaker feesfrom Genzyme-Sanofi, Novartis, GeNeuro and Roche; (iii) research support from Siemens, GeNeuro, Roche. M.A. has received consultancy fees from GSK and Sanofi-Genzyme. P.M. has received support from Biogen and Cliniques universitaires Saint-Luc Fonds de Recherche Clinique. D.S.R. has received research support from Abata, Sanofi-Genzyme, and Vertex. The other authors have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper."}
+{"text": "APOB gene, which traces to the bull Maughlin Storm and was spread to elite show cattle largely through his grandson Braedale Goldwyn. Calves that are homozygous for CD can neither transport dietary fat from the gut nor synthesize low-density lipoproteins, and they perish before 6 mo of age. The mutation may have a partially dominant effect, with lowered cholesterol in heterozygous animals. Our aims were to evaluate serum cholesterol in heterozygous animals, evaluate daily phenotypic records of lactating CD carriers, and determine whether CD carrier status is associated with show ring success. Blood was collected on a single date in May 2018 from 12 carriers and 14 controls and 1 yr later from 17 carriers (7 repeated for 2018) and 27 controls (6 repeated from 2018); animals ranged in age from 14 to 84 mo and varied in stage of lactation and pregnancy status. Serum samples were analyzed for cholesterol concentration (mg/dL) and results were analyzed using mixed models that included linear and quadratic effects of days in milk and days carried calf. Daily phenotypic records of milk yield, milking time, milk conductivity, activity, and body weight were compared between carriers and controls. Additionally, the CD carrier status of cows that were sired by known CD carriers and had available genotypes was recorded. Cows placing in the top 5 and top 10 of their classes at World Dairy Expo between the years of 2006 and 2019 were included in our analysis. Chi-squared tests for equal proportions were evaluated to determine whether the following were associated with CD status: placing in the top 10 with repeated placings from the same cow included, placing in the top 5 with repeated placings, top 10 cows considering individuals only once, and top 5 cows considering individuals only once. Control animals (noncarriers) had significantly higher serum cholesterol (116.21 \u00b1 6.45 mg/dL) than CD carriers (90.15 \u00b1 7.49 mg/dL). There was no difference between carriers and controls in milk yield, milk conductivity, or activity. However, CD carriers had shorter milking times and lower body weights than their herdmates. Further, there were more CD carriers in the top 5 and more carriers tended to place in the top 10 at World Dairy Expo when repeated placings were included. These data support observations that CD is partially dominant and indicate that CD carriers display other phenotypic differences from their herdmates that may give them a small advantage at elite cattle shows.Cholesterol deficiency (CD) in Holstein cattle results from an insertion in the  CD) is a metabolic disorder that causes low blood cholesterol, reduced feed intake, reduced growth, chronic diarrhea, pneumonia, and eventual death in Holstein calves  B; the mutation has been traced to the bull Maughlin Storm (0073HO02012)  . AnimalsHO02012) .APOB was not detectible by standard chips now used for genomic testing, so a haplotype associated with CD (HCD) served as the marker. Most genomic tests now directly include the relevant SNP. In the United States Holstein population, the haplotype frequency is estimated to be 2.5% (https://worlddairyexpo.com/) a record 11 times.At the time of discovery, the causative mutation in  be 2.5% . The freIn CD carriers, total blood cholesterol appears to be reduced compared with that of noncarriers, indicating that the mechanism of inheritance of CD is partially dominant . RecentlOur objectives were to evaluate serum cholesterol levels in CD carriers compared with control herdmates; to investigate differences between mature CD carriers and their noncarrier herdmates in milk yield, milking speed, milk conductivity, activity, and BW; and to determine whether CD carriers have a competitive advantage in the show ring compared with their noncarrier paternal siblings.Genotyped cows and heifers (IACUC protocol #47580) from the Pennsylvania State University Dairy Teaching and Research Center herd (University Park) were candidates for this study. All animals with an HCD status of 1 , HCD status of 3 , and with CD status  were considered CD carriers. Samples from nulliparous (n = 27), primiparous (n = 20), and multiparous animals (n = 23) were included in our data. Carriers were matched with noncarrier controls by age, stage of lactation, and reproductive status.DCC) of pregnant animals was 123 \u00b1 94 (14 CD carriers) and 116 \u00b1 88 (21 controls).In May 2018, blood was collected from 12 CD carriers and 14 noncarrier controls. Blood was again taken in May 2019. Carriers sampled in 2018 that were still in herd were sampled (n = 7), as were controls (n = 6). Additional CD carriers were identified in the herd (n = 10) and controls were age matched (n = 21); we sampled more controls in 2019 to increase the power to detect differences. Samples were from 10 nulliparous CD carriers (age = 21 \u00b1 5 mo) and 17 controls (age = 20 \u00b1 4 mo). Average DIM for lactating cows at the date of sampling was 90 \u00b1 68 (19 CD carriers) and 101 \u00b1 85 (24 controls). The average days carried calf (g within 2 h of collection. Serum samples (n = 70) were stored at \u221220\u00b0C and then thawed and analyzed for cholesterol concentration (mg/dL) using an EzyChrom Cholesterol Assay Kit  per the manufacturer's instructions.Tubes were stored on ice and centrifuged for 10 min at 1,200 \u00d7 Daily milk records and BW were compiled over all lactations during the lifetime of CD carriers (n = 13) and controls (n = 19) that had cholesterol levels analyzed and that had calved. Daily milk weight (kg), milking speed, and milk conductivity were recorded at each milking via an automated management system ; daily BW were collected by the AfiFarm 3.04E scale system when exiting the milking parlor , and activity levels were determined by a pedometer that also served as electronic identification in the milking parlor. Any records missing both milk yield and BW were eliminated from the set. A total of 18,738 observations from calving to 305 DIM were included in our final analysis: 8,306 observations of CD carriers and 10,432 observations of controls.https://worlddairyexpo.com/pages/Dairy-Cattle-Show-All-Results.php). Lactating Holstein cows that placed in the top 10 in the open show from 2006 to 2019 were considered elite show cows. The CD status of their sires was determined using public records available from Holstein Association USA  or the Canadian Dairy Network . A total of 13 sires had daughters with available genotypes from HUSA or CDN with daughters in the top 10 of their respective classes: Maughlin Storm, Braedale Goldwyn, Golden-Oaks St Alexander (007HO08221), Lirr Drew Dempsey (007HO09264), Scientific Destry (094HO13666), Gillette Final Cut (200HO03280), Comestar Lauthority (200HO05588), Carrousel Resurrect-Red (200HO09442), Pursuit September Storm (200HO03067), Comestar Stormatic (200HO04144), Ladino Park Talent (200HO07030), Gillette Windbrook (200HO03501), and Gillette Windhammer (250HO00914).Show results were accessed from online records available on World Dairy Expo's website  with the following model:ijklmny = serum cholesterol concentration (mg/dL); \u03bc = mean; CS = carrier status i (carrier or control); jb = coefficient of regression on DIM and DIM2 nested within lactating status k ; lDCC = days carried calf nested within pregnancy status m ; nCow = the random effect of individual cow nested within carrier status; and \u025b = random error. Least squares means were generated using a Tukey adjustment, with DIM and DCC set to 0. Cholesterol increased from calving to approximately 200 DIM by 105 mg/dL and then declined slowly; in contrast, cholesterol decreased over the progression of gestation (\u221223 mg/dL).Daily phenotypes of milking time, milk conductivity, and activity of CD carriers and controls were analyzed using the MIXED procedure:ijklmny = dependent variable of milking time, milk conductivity, or activity; \u03bc = mean; CS = carrier status i (carrier or control); DIM = days in milk grouped in 2-wk intervals j (1 to \u226522) nested within lactation group (LG) k ; Date = random effect of date of measurement l; Cow = the random effect of individual cow m in lactation number n nested within carrier status; and \u025b = random error. Least squares means were generated using a Tukey adjustment. The model used to analyze daily BW and daily milk yields was the same except that regression on genomic PTA of either BW composite  or milk yield  was included.https://www.R-project.org/) to determine whether the proportions of CD versus noncarrier daughters of the eligible sires differed from equality. Four tests were performed to determine whether show ring success was associated with CD carrier status: placing in the top 10 with cows allowed to contribute multiple years, placing in the top 5 with cows allowed to contribute multiple years, top 10 cows only considering individuals once, and top 5 cows only considering individuals once.For the elite show cow analysis, chi-squared tests for equal proportions were conducted using the stats package in R . The intraclass correlation coefficient of the model was 0.3355 for animals with repeated measures of serum cholesterol. P > 0.05). Lactating CD carriers were, on average, 33.44 kg lighter than controls (P < 0.05) and milked out about 1 min faster than controls (P < 0.01). Cholesterol deficiency status was significantly associated with lower daily BW, even after accounting for genomic PTA of BW composite . It has yet to be determined if this is because carriers carry less fat or because they were smaller in frame size and stature. Carriers of CD could carry less condition as it is known that higher circulating APOB levels are positively correlated with central fat distribution in humans  from an expected frequency of 115.5 CD carriers and 115.5 non-CD carriers if CD status is unrelated to show ring success. Because some daughters were shown and placed in the top 10 multiple times, the 231 instances represented results from 135 daughters of the CD carrier sires; of these, 93 were Braedale Goldwyn daughters. If each daughter was only counted once, there were 72 carriers and 63 noncarriers, which did not differ significantly from expectation. In the top 5 placings, there were 135 instances (82 CD carriers and 55 noncarriers) comprised of 84 individual cows (48 CD carriers and 36 noncarriers). Carriers were more heavily represented in the top 5 of their class and significantly (P < 0.05) more than noncarriers when repeated wins were considered. Cholesterol is one of the main lipids of bovine skin, and \u201cthin, loose, and pliable\u201d skin is preferred in type evaluations (The results of the chi-squared analysis are shown in luations . HoweverAPOB mutation are not at a disadvantage when evaluated for type and may carry an advantage in elite type competitions.Serum cholesterol levels of cattle that carry CD were lower than those of their noncarrier herdmates. Carriers also had faster milking speeds and lower BW than their herdmates, but results were based on a limited sample of carriers. Elite show cows that carry the"}
+{"text": "Natural killer (NK) cells are a part of innate immunity that can be activated rapidly in response to malignant transformed cells without prior sensitization. Engineering NK cells to express chimeric antigen receptors (CARs) allows them to be directed against corresponding target tumor antigens. CAR-NK cells are regarded as a promising candidate for cellular immunotherapy alternatives to conventional CAR-T cells, due to the relatively low risk of graft-versus-host disease and safer clinical profile. Human induced pluripotent stem cells (iPSCs) are a promising renewable cell source of clinical NK cells. In the present study, we successfully introduced a third-generation CAR targeting CD19, which was validated to have effective signaling domains suitable for NK cells, into umbilical cord blood NK-derived iPSCs, followed by a single-cell clone selection and thorough iPSC characterization. The established single-cell clone of CAR19-NK/iPSCs, which is highly desirable for clinical application, can be differentiated using serum- and feeder-free protocols into functional CAR19-iNK-like cells with improved anti-tumor activity against CD19-positive hematologic cancer cells when compared with wild-type (WT)-iNK-like cells. With the feasibility of being an alternative source for off-the-shelf CAR-NK cells, a library of single-cell clones of CAR-engineered NK/iPSCs targeting different tumor antigens may be created for future clinical application. Cancer has been the second leading cause of death globally for many years and is attributable to one in six deaths. By 2040, the global burden of cancer is projected to be 28.4\u2009million new cases, a 47% rise from 2020 . The maiNatural killer (NK) cells are part of the first line of defense in the innate immunity, with a potent ability to kill malignant transformed cells and tumor cells without prior sensitization. NK cell-mediated cytotoxicity depends on the balance between signals from activating receptors, e.g., CD16, natural cytotoxicity receptor family members , killer immunoglobulin-like receptors (KIRs), and NKG2D, and inhibitory receptors . Trials The breakthrough in human induced pluripotent stem cell (iPSC) research has opened up vast opportunities for regenerative medicine. iPSCs are an ideal starting material for next-generation CAR-related immune cell therapy, due to the ease of genetic manipulation and limitless expansion during the iPSC stage ,12. Curr\u2212CD56+CD16+ NK cells isolated from UCB from a healthy newborn by using non-integrable episomal reprogramming [TP53 and an extra plasmid encoding transient EBNA-1, which enhanced the reprogramming efficiency [Previously, we generated an NK/iPSC line from CD3ficiency , were inficiency , we tranficiency . After tficiency , and scrOCT4, NANOG, and SOX2) in comparison to H1 ESC and wild-type (WT)-NK/iPSCs , mesoderm , and endoderm (LEFTY1 and AFP) in EBs on day 14 of differentiation when compared to non-differentiated cells. Immunofluorescence showing the expression of markers for endoderm , mesoderm , and ectoderm (Nestin) further confirmed the in vitro differentiation  C, flow cNK/iPSCs E. The loNK/iPSCs F. TransdNK/iPSCs G. Short NK/iPSCs H. For a NK/iPSCs A. Figurentiation C.+ HPCs into NK cells, with slight modifications, as schematically depicted in +, mostly with CD43+, thus confirming that they were HPCs. We observed a decrease in CD34+ floating cells on day 20 onwards, indicating that HPCs started to differentiate into more mature cells in the NK lineage. On days 34 and 41 of culture, the iPS sacs completely disappeared, while free-floating, multicellular aggregates were observed -derived REH cells and human Burkitt\u2019s lymphoma (BL)-derived Raji cells. REH and Raji cells, so-called target cells (T), were labeled with PKH67 green fluorescence and cocultured with CAR19-iNK-like or WT-NK/iPSC-derived NK-like (WT-iNK-like) cells, so-called effector cells (E), at E:T ratios of 1:10, 1:5, 1:2, and 1:1 for 4 h, as schematically depicted in To confirm that the anti-tumor activity of CAR19-iNK-like cells was mediated via NK signaling, we measured NK cell activation markers, including CD161, CD158, and Nkp30, upon exposure of the cells to PKH67-labeled target tumor cells using the gating strategy shown in NK cell-based immunotherapy, including that of CAR-NK cells, has been used in several clinical trials, due to its potent ability to kill tumor cells with a low risk of major complications such as GvHD, acute CRS, and neurotoxicity. Herein, we were able to introduce the third-generation CAR, targeting CD19, which was validated to have effective signaling domains suitable for NK cells, in human NK-derived iPSCs to subsequently generate anti-CD19 CAR-NK-like cells, referred to as CAR19-iNK-like cells.CAR-NK cells have constituted a promising area of cellular immunotherapy innovation, particularly allogeneic CAR-NK cells, which are recognized as a promising off-the-shelf product. The majority of ongoing clinical trials involve CAR-NK-92 cells, which modified the FDA-approved NK-92 cell line, for treatment of both hematologic cancers and solid tumors ,24. EarlRecently, iPSCs have represented an attractive cell source for producing a large number of NK cells due to the limitless expansion during the iPSC stage ,28,29. PO-GlcNAcylation, a nutrient-sensitive posttranslational modification of proteins, in HPC differentiation from iPSCs, as we previously found that O-GlcNAcylation is a key determinant of hematopoietic stem cell (HSC) fate decision and certain lineage-specific differentiation processes, including megakaryopoiesis [It is important to note that we may further improve NK cell differentiation by using different approaches to improve the differentiation efficiency at the stage of HPC differentiation via mesoderm induction and/or NK commitment. Previous studies have demonstrated that bone morphogenetic protein 4 (BMP4), fibroblast growth factor 2 (FGF2), WNT/\u03b2-catenin, and Activin/Nodal play critical roles for mesoderm formation ,35,36,37opoiesis , erythroopoiesis , and denopoiesis . For imp2.Human iPSCs, MUSIi013-A single-cell clone, reprogrammed from UCB-derived NK cells (NK/iPSC)  were rouLentiviral production was performed using HEK293FT packaging cells in conjunction with pCMV.dR8.2 dvpr lentiviral packaging and pCMV-VSV-G envelope plasmids (Addgene #8454 and #8455) . BrieflyMUSIi013-A cells were transduced with lentiviral particles carrying the anti-CD19 in the presence of 4 \u03bcg/mL hexadimethrine bromide . Single cells were seeded on irradiated human foreskin fibroblasts in NutriStem medium supplemented with SMC4 small-molecule cocktail inhibitors containing PD0325901 , CHIR99021, Thiazovivin, and SB431542  [TM Terminator Cycle Sequencing Kit v3.1 .Genomic DNA was isolated using a PureLink Genomic DNA Mini Kit . The target regions for DNA sequencing were amplified by PCR using Q5 High-Fidelity DNA Polymerase  with specific primers, and the resulting PCR products were purified by a GenepHlow Gel/PCR kit . A total of 0.2 \u03bcg PCR product was then used for DNA sequencing using ABI PRISM BigDyeCAR expression in iPSCs was determined by flow cytometry based on Fab fragments. Cells were incubated with FITC-conjugated anti-mouse-IgG, F(ab\u2032)2 fragment antibody (F(ab\u2032)2-FITC; Jackson ImmunoResearch, West Grove, PA, USA) for 30 min at 4 \u00b0C and analyzed using a BD FACS Canto .CAR expression in the differentiated cells was evaluated by target antigen-based detection to ascertain its binding activity. Briefly, cells were incubated with 10 \u03bcg/mL rhCD19 (20\u2013291) protein with His tag to the C-terminus  for 1 h at 4 \u00b0C, followed by an incubation with FITC-conjugated anti-His tag antibody  for 15 min at room temperature and flow cytometric analysis. Cells that were incubated with His tag-FITC, but not with rhCD19, were used as a basal control. The percentage of cells that expressed CAR could be calculated from the subtraction of FITC-positive cells in the basal control from those with the target protein.Cells were fixed in 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100/PBS for 10 min, and blocked with 3% BSA/PBS for 1 h. The cells were incubated with primary antibodies in 1% BSA/PBS overnight at 4 \u00b0C, followed by secondary antibodies at room temperature for 1 h. Nuclei were counterstained with Hoechst 33342 (Thermo Fisher Scientific) for 30 min at room temperature and visualized under fluorescence microscope  with NIS-Elements D Software .TM Select (Gibco), blocked with 10% human AB serum, stained with Alexa Fluor 488-SSEA-3 and Alexa Fluor 647-TRA-1-60 , and analyzed by BD FACS Canto flow cytometer (BD Biosciences) with FlowJo software (V10.4.1). For phenotypic analysis of HPC induction and NK commitment, the cells were resuspended in FACS buffer , incubated with antibody cocktail for 30 min at 4 \u00b0C in the dark, and analyzed by BD FACS Canto flow cytometer. The antibodies used in this study included FITC-CD16, FITC-Nkp46, PE-CD56, PE-Nkp44, PerCP-CD45, PerCP/Cy5.5-CD94, APC-CD34, PE/Cy7-CD43, PE/Cy7-KIR, Alexa Fluor 700-CD161, and BV605-Nkp30 (BioLegend).For analysis of pluripotency markers, iPSCs were dissociated into single cells using TrypLE\u00ae  and converted to complementary DNA using the RevertAid First Strand cDNA synthesis kit (Thermo Fisher Scientific). The qPCR reactions were performed on the CFX384 Touch Real-Time PCR detection system  using SYBRTM Select Master Mix (Thermo Fisher Scientific). The cycle parameters started with an activation step at 95 \u00b0C for 2 min, followed by 40 cycles of denaturation at 95 \u00b0C for 15 s and annealing/extension at 60 \u00b0C for 1 min.Total RNA was isolated using TRI ReagentThe standard G-banded karyotyping was performed at the Department of Obstetrics and Gynecology, Faculty of Medicine Siriraj Hospital, Mahidol University. A total of 25 metaphases at a band resolution of 400\u2013450 were analyzed.STR analysis was performed at the Department of Forensic Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University. A total of 16 loci were tested.TM, 0.1 mM MEM non-essential amino acid, 0.1 mM \u03b2-mercaptoethanol, 1\u00d7 insulin-transferrin-selenium-ethanolamine, and 100 U/mL penicillin/streptomycin (Gibco). The medium was replaced every other day. On day 7, EBs were transferred onto a 0.1% gelatin-coated plate and cultured at 37 \u00b0C and 5% CO2 for another 3 weeks.The iPSCs were harvested into small clumps using 1 mg/mL Dispase (Gibco) and cultured on low attachment dishes in knockout DMEM supplemented with 20% knockout serum replacement, 2 mM GlutaMAXTM (Gibco). The HPC induction protocol was slightly modified from the protocol used by Lupo KB, et al. [g for 5 min to promote EB formation and incubated for 6 days, during which the medium was changed every 3 days by removing 70 \u03bcL of medium from each well and adding 100 \u03bcL of freshly prepared medium without ROCK inhibitor.iPSCs were pre-treated with ROCK inhibitor for 1 h before single cell dissociation by Accutase, et al.  by meansOn day 6, 30 EBs were transferred onto a Matrigel-coated 6-well plate in 4 mL NK cell differentiation medium consisting of STEMdiff APEL2 medium, 20 ng/mL SCF, 20 ng/mL IL-7, 10 ng/mL IL-15, and 10 ng/mL Flt3 ligand, which was supplemented with 5 ng/mL IL-3 for the first week. The medium was half-changed every two to three days for 3\u20134 weeks.2+-rich buffer for 15 min at room temperature. Samples were analyzed immediately via FACS analysis. Total cell death of target tumor cells was defined as annexin-V and/or 7-AAD-positive cells in the PKH67-positive cell population.Target tumor cells were labeled with PKH67 lipophilic dye for 5 min at 37 \u00b0C and were subsequently incubated with the corresponding ratio of effector CAR19- and WT-iNK-like cells in a total concentration of 20,000 cells per well for 4 h at 37 \u00b0C in a round-bottom 96-well plate. After that, the cell mixture was harvested and stained with PE-conjugated annexin-V and 7-AAD  in Cap-value < 0.05 considered to be significant. Data represent mean \u00b1 SD or mean \u00b1 SEM from three or more independent experiments as indicated. Statistical analysis between two groups was performed by Student\u2019s unpaired t-test at a significance level of p < 0.05.Prism 9  was used for all data and statistical analysis, with a In summary, our findings reveal a promising CAR design and production protocol for the establishment of a single-cell clone of CAR-engineered NK/iPSCs, which is highly desirable for clinical application. We also showed its feasibility to be differentiated into CAR-iNK-like cells, which displayed high and selective cytotoxicity toward corresponding leukemia and lymphoma cells in vitro, using serum- and feeder-free protocols. Further studies should focus on improving differentiation efficiency at the stages of HPC induction and/or NK commitment, as well as an expansion of differentiated CAR-iNK-like cells to obtain a sufficient number of cells for clinical applications."}
+{"text": "Stenotus rubrovittatus (order Heteroptera: family Miridae), is a notorious insect pest in Japan that causes pecky rice. In the present study, we sampled this insect pest in the northern part of Honshu Island in Japan and investigated its associated microbiota. The results obtained showed that Pantoea dominated the associated microbiota and was the sole genus detected in all samples. The dominant Pantoea were phylogenetically close to rice pathogens. The present results suggest that the sorghum plant bug needs to be regarded and controlled not only as a notorious insect pest, but also as a potential vector of rice pathogenic Pantoea spp.The sorghum plant bug,  Insect-microbe symbiotic interactions are ubiquitous in nature . Insect-Stenotus rubrovittatus (order Heteroptera: family Miridae) (Rice is a major and important crop in Japan and Asia. The sorghum plant bug Miridae) A is a noS. binotatus, \u201cRickettsia-like symbionts\u201d were found in the nuclei and cytoplasm of some epithelial cells of the digestive tract using transmission electronic microscopy  consists of versatile and diverse species and includes plant pathogenic species, clinical and environmental isolates, and symbiotic species of various insects, particularly pentatomid stink bugs  (Supplementary Insect sampling was performed at 24 locations over seven prefectures (29 samples) in the northern part of Honshu Island in Japan  were subjected to quality filtering, resulting in 16,501\u201363,999  qualified and merged amplicon sequences per sample   . \u201cUnclasominated . No sequ3 to 8.0\u00d7106  .The size of the bacterial population associated with the sorghum plant bug was estimated by performing quantitative PCR targeting the bacterial 16S rRNA gene. The estimated size varied among samples from 2.9\u00d710Pantoea dominated the associated microbiota and was the sole genus detected in all samples. Previous studies reported that some pentatomid stink bugs, e.g., the brown-winged green stink bug Plautia stali and the sloe bug Dolycoris baccarum, develop midgut crypts, the lumen of which is colonized by Pantoea symbionts to establish obligate symbiosis . Microbes Environ 38: ME22110.Sato, Y., Akao, T., and Takeshita, K. (2023) High Prevalence of https://doi.org/10.1264/jsme2.ME22110Supplementary Material"}
+{"text": "This retrospective study analyzed breathing patterns and age subgroups effect on cortical bone quality of the mandible in growing subjects, aiming to explore the application value of facial skeletal pattern combined with cortical bone density detection in early screening diagnosis of mouth breathing.One hundred twenty-six participants were divided into four groups: mouth breathing group  and nasal breathing group . The mandibular anterior, middle, and posterior cortical bone mineral density (CBMD), cortical bone width (MCW), ANB, and FMA values were measured. Independent T-test and Mann\u2013Whitney U test were used to compare the measured values. Binary logistic regression was employed to analyze the correlation between measured variables and the children\u2019s breathing patterns. ROC analysis was used to determine the ability of the cortical bone density measurements in early screening diagnosis of MB.Mouth breathing had a negative impact on CBMD and MCW of the pre-mandibular (Pog) in subjects aged 7\u20139\u00a0years and also impacted the development of (Pog) and submandibular (Me) sites in subjects aged 10\u201312\u00a0years. Older children in the nasal breathing group have higher CBMD, MCW, and SNB values and lower FMA values. Single-factor and multiple-factor logistic binary regression analysis showed that FMA, MSPogCBMD, MSPogMCW, and ANB are correlated factors for children at risk of mouth breathing.Mouth breathing pattern is closely associated with decreased mandibular CBMD and MCW values in children aged 7\u201312, where the anterior (Pog) and inferior (Me) sites of anterior mandible are more significantly affected. Furthermore, in combination with facial skeletal pattern, it provides a basis for the early warning diagnosis of mouth breathing. The growth of the craniofacial region involves significant changes in the facial dimension. Among the craniofacial structures, the mandible plays a crucial role in maintaining proper facial appearance and function, and any abnormalities in mandibular development can lead to various dental and functional problems, such as malocclusion, sleep apnea, and facial asymmetry , 2. TherPartial obstruction of the airways frequently due to chronic adenoid hypertrophy leads to mouth breathing and altered oral muscle activity, resulting in the posterior rotation of the mandible, causing an increased overjet. Simultaneously, this restricted mandibular rotation can contribute to a skeletal open bite , 4. ConvA study performed by Eimar et al. investigated the mandibular bone quality in children using panoramic radiographs and reported that the altered normal breathing was associated with significantly reduced mandibular cortical width compared to typically nasal breathing individuals . AnotherFractal dimension analysis offers a mathematical approach for assessing the complexity and irregularity of bone structures. A radiation-free alternative to objectively quantifying bone density reported by Jurczyszyn et al. , which cTherefore, we aim to evaluate the cortical bone mass of the mandible in children aged 7\u201312 with different breathing patterns using CBCT data.The Stomatological Hospital of Xi\u2019an Jiaotong University Ethics Committee has approved this study. Ethical approval number: Xjkqll [2018] No.17.Using a formula proposed by Pandis , the samIn this investigation, 126 CBCT scans were gathered from 126 individuals   cone beam machine at 120 kV, 5 mA, 14\u2009\u00d7\u200917 cm FOV, 0.4 mm voxel, with a scan duration of 8.9 s. The CBCT images were then saved in DICOM format\u00a0.A multidisciplinary team consisting of an orthodontist and an ENT specialist evaluated the diagnostic criteria for respiratory patterns. First, the orthodontist screens for mouth respiration, which includes (1) Asking parents to record a video, checking whether the child\u2019s lips are closed after falling asleep, whether snoring can be heard, and whether there are symptoms of drooling on the pillow, (2) Asking if the child is prone to fatigue, allergies, and colds, (3) Clinical examination of the child\u2019s habitual lip posture, nostril shape, and size and perform Glatzel mirror test . All subThe DICOM files were imported for measurements using Dolphin Imaging software  version 11.7. The axial plane of the CBCT images had been aligned with the Frankfort horizontal\u00a0plane (FHP), the midsagittal plane was matched with the patient\u2019s midline at Nasion (N) point, and the coronal plane had been modified to be perpendicular to the axial plane and passing through Porion point Fig.\u00a0. One invIn the initially aligned CBCT images, the cross sections of the anterior, middle, and posterior segments of each subject\u2019s mandible were selected through the following steps: 1. Mandibular symphysis, chosen at the Sagittal plane section images of the anterior mandible Fig.\u00a0A; 2. MidMCW values were measured as the linear distance at each planned selected area. For mandibular symphysis (MS), the MCW was measured at pogonion (Pog), Menton (Me), and genial tubercle (Ge) areas. For the middle of the mandible, the MCW values were measured at one location, which is inferior or basal cortical bone width (IMF). While for the posterior mandible, MCW values were measured at buccal (B6), lingual (L6), and inferior areas (I6) Table . All MCWAll measurements were analyzed using SPSS software . The Shapiro\u2013Wilk test was used to test the normality of the variables. Twenty CBCT images were randomly selected to investigate the reliability of the measurement method used. The two researchers repeated all the measurements one week after the first attempt. Inter-investigator and intra-investigator errors were evaluated using intra-group correlation coefficient tests to calculate measurement errors.The 126 participants were grouped according to age and breathing patterns. Mandibular CBMD and MCW values conforming to normal distribution were expressed as mean\u2009\u00b1\u2009standard deviation (\u00b1\u2009s), and independent sample t-test was used. Parameters that did not conform to the normal distribution were represented by the median (quartile distance) (M(IQR)). The Mann\u2013Whitney test was used to compare the differences in CBMD and CWM among different breathing patterns and age groups. Mouth respiration (binary outcome index) was used as the dependent variable; age, ANB, SNA, SNB, FMA, CBMD, and MCW at each site were used as covariates for single-factor screening. The logistic regression model was established by \u201centry\u201d method, and the screening criteria were \u03b1_in\u2009=\u20090.05 and \u03b1_out\u2009=\u20090.10. Sites with statistically significant results were selected as predictors by binary logistic regression . The regression equation is established. Receiver operating characteristic curve (ROC) was plotted.The results of the (ICC) test showed consistency in measurements within the same researcher (0.90\u20140.95) and between researchers (0.89\u20140.95), indicating good reliability of the measurement method used. The Shapiro\u2013Wilk test results indicated that the  values were normally distributed where independent sample t-test was used to determine the significant difference. In contrast, the Mann\u2013Whitney U test was used for the remaining sites with non-normally distributed variables. Scatter dot plots with mean, standard deviation, and significance were constructed based on the results of the independent samples t-test and the Mann\u2013Whitney U test for CBMD, and MCW values between the different breathing patterns within the same age groups and different age groups Figs.\u00a0.Fig. 6CBP\u2009=\u20090.028, 0.001,0.0001) showed significant differences; while in the 10\u201312 years age group, MSPogCBMD, MSMeCBMD, MSPogMCW, FMA and MSMeMCW  were differed significantly. The CBMD and MCW values were higher, and FMA values were lower in nasal breathers compared to mouth breather individuals.When comparing the CBMD and MCW values between the mouth and nasal breathers within the same age group, significant differences were found as follows: in the 7\u20139 years age group, MSPogCBMD\u3001MSPogMCW\u3001FMA . While in the mouth breather with different age groups, MSPogCBMD, MSMeCBMD, MSPogMCW, IMFMCW, B6MCW, I6MCW  showed statistically significant differences. In the 10\u201312 age group, CBMD, MCW, and SNB values were higher, and FMA was lower than that found in the 7\u20139 age group. The number of significantly different research sites in terms of CBMD and MCW was more in the nasal breathers group (10 sites) compared to the mouth breathers group (6 sites) ; considering that the negative \u03b2 value indicated decreased likelihood of falling into MB group, while the positive \u03b2 value indicated increased likelihood of falling into NB group (Table The regression equation was formulated as logit(Y)\u2009=\u2009-21.537\u2009+\u20090.002*X1-1.438*X2\u2009+\u20090.723*X3\u2009+\u20090.27*X4  MSPogMCW was the most infuencing factor, followed by FMA, ANB, and MSPogCBMD  and mandibular cortical width (MCW) at different age groups. Among 7\u20139-year-olds, CBMDPog exhibited a substantial increase of approximately 9.2%, and MCW Pog demonstrated an even more pronounced augmentation, with values approximately 31.6% higher in nasal breathers compared to mouth breathers, highlighting the influence of respiratory patterns on craniofacial development at the anterior chin point (Pog). Similarly, notable differences were observed in the 10\u201312-year-old group in CBMD and MCW values. CBMD at Pog showed a significant increase of about 15.5% in nasal breathers, while MCW exhibited a substantial rise of approximately 35%. At the Menton point, CBMD displayed a significant 30% increase, and MCW demonstrated an approximately 32.6% rise in nasal breathers compared to their mouth-breathing counterparts. These findings underscore the importance of nasal breathing in fostering improved CBMD and MCW at specific mandibular points in different age groups, highlighting the potential impact of respiratory patterns on craniofacial development.The analysis suggests this may be related to intermittent hypoxia (IH) caused by mouth breathing which can affect mandibular bone development. IH can lead to sleep disorders that cause changes in hormone levels and disruptions in melatonin secretion, a known regulator of bone mass. The sympathetic nervous system (SNS) coordinates various physiological functions, including the sleep/wake cycle. Sleep disorders can lead to excessive SNS activity, resulting in decreased bone mass. Hong et al. have demonstrated that activation of \u03b22-adrenergic receptors during growth may induce delayed mandibular bone growth in rats with IH . SwansonThe relationship between malocclusion and breathing patterns is an interrelated phenomenon. Malocclusion can contribute to improper breathing patterns, and conversely, improper breathing patterns can exacerbate malocclusion . Our stuMalocclusions are multifaceted conditions that affect dental and craniofacial structures and can also participate in temporomandibular disorders (TMD) development. Boening et al.  sheds liAnimal experiments have also demonstrated a correlation between mouth breathing, delayed mandibular bone metabolism, and low mandibular bone density. Raff et al. and Song et al. reported that bone density decreased in rats exposed to a low oxygen environment , 23. KimIn addition to hypoxia, abnormal muscle movements accompanying oral breathing patterns are associated with adaptive jaw shape and density changes. According to the Moss theory, skeletal muscle contractions are typical loading events for the functional matrix of bone . This prOur study found that the nasal breathing pattern in the 10\u201312-year-old group was associated with a higher prevalence of a balanced facial profile. In comparison, the 7\u20139-year-old group had a higher prevalence of high-angle facial profiles. This may be related to dietary habits in children, where younger children tend to consume softer and more easily digestible food . ChildreOur research highlights the critical relationship between respiratory patterns and craniofacial development. Acknowledging that the upper airway volume plays a pivotal role in this complex interplay is important. Dastan et al.  has inveFurthermore, we have proposed a method that combines sagittal and vertical bone profiles with cortical bone width and density to provide an objective evaluation index for the initial screening and classification of mouth and nasal breathing patterns in children. Experimental results showed that under the same conditions, the accuracy rate of breathing pattern inference of the binary logistic correlation model was 85.7%, surpassing the single data source method. In addition to validating the existing symptoms in children with mouth breathing patterns, this study further explored other factors related to craniofacial manifestations, providing secondary prevention evidence for the early detection, diagnosis, and treatment of oral breathing system disorders.Bone metabolism is a complex process influenced by various factors such as dietary habits, the severity of mouth breathing disorders, and the duration of the condition. Due to the limited sample size, the study did not include children with skeletal Class III malocclusion. Future studies should increase sample sizes and employ rigorous experimental designs and prospective research approaches to further investigate these relationships. Additionally, it would be beneficial to establish reference datasets representing average jawbone quality for different regions, ages, genders, and ethnicities. Comparing bone tissues from stable craniofacial or body structure regions as control variables could also help validate the relationship between low bone density, skeletal patterns, and breathing patterns.The oral respiratory pattern may lead to the decreased trend of mandibular CBMD and MCW in children aged 7\u201312 years, and the Pog and Me sites in the lower anterior mandible are more significantly affected. The combined diagnosis with bone facial pattern variables provides a certain basis for the early warning of oral respiratory pattern."}
+{"text": "This study aimed to compare the performance of Ultrasonography (US) and Magnetic Resonance Imaging (MRI) in assessing the Lateral Periarticular Space (LPAS) of Temporomandibular Joints (TMJs) in patients with Juvenile Idiopathic Arthritis (JIA).U test. Correlation and agreement between MRI and US measurements in JIA group\u00a0were tested using Spearman rank correlation and Bland\u2013Altman method.The LPAS width was evaluated in two different patient groups. In the JIA group, including 29 children (13\u2009\u00b1\u20092.8\u00a0years) with JIA, the LPAS width was measured with both MRI and US. In the healthy group, including 28 healthy children (12.6\u2009\u00b1\u20092.5\u00a0years), the LPAS width was measured only with US. Comparisons of LPAS width based on patient groups and TMJ contrast enhancement in MRI were evaluated by applying the Mann\u2013Whitney The LPAS width was significantly greater in the JIA group than in the healthy group. In the JIA group, the LPAS width was significantly greater in TMJs with moderate/severe enhancement than those with mild enhancement. A positive significant correlation between MRI and US measurements of LPAS width was found in the JIA group. In the same group, Bland\u2013Altman method showed a good level of agreement between MRI and US measurements.Although, US cannot replace MRI in the evaluation of TMJ in patients with JIA, US could be used as a supplementary imaging method to MRI in assessing the TMJ disease. Temporomandibular Joints (TMJs) are involved in 17\u201387% of patients affected by Juvenile Idiopathic Arthritis (JIA) depending on subtype, diagnostic criteria used, and ethnicity [Joint involvement is characterized by episodes of active inflammatory arthritis leading to chronically progressive joint destruction. Further, JIA occurs mainly in the developmental age, and the presence of the disease at a temporomandibular level may severely compromise mandibular growth \u201311.For this reason, an early diagnosis of TMJ involvement and a continuous follow-up are needed to reduce the TMJ damage and improve the quality of life in patients with JIA. This approach has the aim of monitoring the progress of the TMJ disease and modulating the therapeutic plan over time.In active TMJ arthritis, pain is a rare symptom and very often children present with normal clinical examination findings , 12.Currently, the heterogeneity of the published studies did not allow to find any clinical outcome measure that can be considered as predictor of TMJ involvement .Clinical assessment is often insufficient for an early diagnosis of the TMJ involvement , 15. TheCE-MRI remains the gold standard for the evaluation of temporomandibular\u00a0disease\u00a0(TMD), given its capability to recognize the signs of the active  and chronic  phases of the JIA at the TMJ level \u201320. HoweAlthough MRI provides the best TMJ analysis due to its high-contrast resolution, ultrasonography (US) can provide complementary information on both condylar morphology and periarticular soft tissue. Therefore, US could be used to assess the TMJ involvement in JIA patients. However, its role in diagnosing arthritis of the TMJ is unclear. Some studies showed no correlation between MRI and US findings and concluded that MRI is the most appropriate imaging technique for detecting TMD in JIA patients , 22. PowThis study aimed to evaluate whether US, compared with MRI, can be considered a suitable imaging technique to assess active TMJ disease in patients with JIA.This study was conducted according to the ethical principles for medical research. It was approved by our local ethics committee . All children and parents were informed about the study protocol. Informed consent was obtained from all participants.The study group (JIA group) included children diagnosed with JIA using the International League of Associations for Rheumatology (ILAR) 2001 diagnostic criteria . The chiThe patients were recruited at our institution by the Dental Clinic, and they were referred to our centre by the Pediatrics Clinic.The control group  consisted of healthy children that matched the patients with JIA for sex and age. In addition, they had healthy TMJs characterized by the absence of TMD or TMJ trauma, absence of rheumatic or systemic disease, and no pharmacological treatment. The healthy controls were recruited from the Dental Clinic.Both groups have no previous or in progress gnathological or orthodontic treatments.The JIA and healthy groups included the patients that were enrolled in a previous pilot study .In this study, the lateral periarticular space (LPAS) width in patients with JIA and active TMJ disease were measured with US. In JIA group, the US measurements of the LPAS width were compared with the ones calculated in the correspondent CE-MRI images in order to identify the level of agreement. The time interval between the two imaging methods (US and CE-MRI) was at most 1\u00a0week. In JIA group, the periarticular TMJ enhancement on CE-MRI was also assessed and classified as mild, moderate/severe.The LPAS width of TMJs was also measured in healthy children by means of US. The healthy group was included to obtain the normal range for LPAS width in children and compared it with the LPAS values found in the\u00a0JIA group.The LPAS contains articular cavity with synovial liquid, articular disc if it is displaced, synovium and capsule. We considered the LPAS width, because the US examination is able to assess only the lateral area of the TMJ. In addition, we used the LPAS, because previous studies have demonstrated the presence of a correlation between US measurements of LPAS width and MRI findings of active inflammation such as joint effusion, synovial thickening, and joint enhancement , 25. TheUS and the CE-MRI of TMJs were performed and assessed by the same expert radiologist (A.B.), with 15\u00a0years of experience in dentomaxillofacial radiology. The same radiologist performed the measurements of LPAS both on US images and on CE-MRI images.LPAS measurements, performed both in US and CE-MRI, were repeated twice by the same radiologist in 15 randomly selected patients after a wash-out period of 1\u00a0week in order to evaluate the intraobserver agreement.On CE-MRI images, the LPAS width and the periarticular TMJ enhancement were evaluated using the department\u2019s picture archiving and communication system .All MRI examinations were performed using a 1.5\u00a0T MRI scanner . MRI examinations were obtained before and after intravenous injection of a gadolinium-based contrast medium. The postcontrast MRI images were acquired on the axial plane with a T1-weighted fat saturated sequence .The LPAS width was assessed on postcontrast axial images at the level of the largest cross-sectional area of the mandibular condyle. The LPAS measurement was obtained using as a reference point the most lateral point of the condylar cortex until the lateral limit of the periarticular tissue Fig.\u00a0.Fig. 1AxAll US examinations were performed with a US machine  using a probe with a frequency of 15\u00a0MHz.The US images were obtained with patient in a supine position with the head turned to the left to assess the right TMJ and to the right to assess the left TMJ, in accordance with Emshoff, Jank . The US The width of LPAS was measured orthogonally from the lateral cortical plate of the mandibular condyle to the outline of the capsule on coronal US images  Fig.\u00a02)2).Fig. 2The distribution of continuous variables was tested using the Shapiro\u2013Wilk test. Normally distributed data were presented as mean and standard deviation, whereas non-normally distributed data were presented as median and interquartile ranges (IQRs). Categorical variables were reported as relative frequencies and percentages.U test. In the JIA group, the Mann\u2013Whitney U test was also used to verify differences of LPAS width between TMJs with mild enhancement and those with moderate or severe enhancement on CE-MRI images.The US and MRI measurements of LPAS width were not normally distributed; therefore, nonparametric tests were used. Comparisons of US and MRI measurements of LPAS between the JIA and the healthy groups were performed by applying the Mann\u2013Whitney Bland\u2013Altman method was applied to assess the agreement between two LPAS consecutive repeated measures (obtained both with US and MRI) and the agreement between MRI and US measurements of LPAS. Correlations between MRI and US values of LPAS was assessed by means of the Spearman\u2019s correlation coefficient.p\u2009<\u20090.05). STATA17  and MedCal  were used to perform statistical analyses.The statistically significant level was set at 5%  females and 3 (10.3) males. 27/29 (93.1%) JIA patients presented bilateral TMJ active disease, whereas the remaining 2/29 (6.9%) JIA patients showed only one-sided TMJ disease. Therefore, an overall of 56 TMJs were considered in the JIA group.The healthy group included 56 TMJs of 28 healthy children, 24 (85.7) girls and 4 14.3%) boys. Patients\u2019 characteristics, clinical and MRI findings at the time of the examinations are listed in Table 4.3% boysp\u2009<\u20090.0001).In the JIA group, the median width of LPAS measured on CE-MRI was 1.3\u00a0mm , whereas on US was 1.0 . In the healthy group, the median width of LPAS measured on US was 0.6\u00a0mm . Based on these findings, the LPAS width measured on US was significantly greater in the JIA group than in the healthy group (p\u2009\u2264\u20090.0133 for both US and CE-MRI).In the JIA group, 15/56 (26.8%) TMJs showed moderate/severe enhancement and 41/56 (73.2%) TMJs showed mild enhancement. In TMJs with moderate/severe enhancement, the median width of LPAS measured on CE-MRI was 1.4\u00a0mm , whereas on US was 1.1\u00a0mm . In TMJs with mild enhancement, the median width of LPAS measured on CE-MRI was 1.2\u00a0mm , whereas on US was 0.9\u00a0mm . Based on these data, the LPAS width was significantly greater in TMJs with moderate/severe enhancement than those with mild enhancement ; the 95% confidence interval (CI) of the lower limit of the agreement ranged from \u2212\u20090.23 to \u2212\u20090.14\u00a0mm, and the 95% CI of the upper limit of agreement ranged from 0.17 to 0.27\u00a0mm ; the 95% confidence interval (CI) of the lower limit of the agreement ranged from \u2212\u20090.30 to \u2212\u20090.19\u00a0mm, and the 95% CI of the upper limit of agreement ranged from 0.17 to 0.28\u00a0mm ; the 95% confidence interval (CI) of the lower limit of the agreement ranged from \u2212\u20090.83 to \u2212\u20090.40\u00a0mm, and the 95% CI of the upper limit of agreement ranged from 0.99 to 1.42\u00a0mm   in US and MRI. Most studies considered effusion as an essential factor for diagnosing of active TMJ arthritis in patients with JIA , 22, 24.Power Doppler US was used to evaluate synovial vascularity in only one study . HoweverThe US LPAS measures, which were performed in the present cohort of patients with JIA and established TMJ active arthritis by CE-MRI, showed a mean of 1.036\u00a0mm with a range of 0.6\u20131.5\u00a0mm. This mean value is lower compared with the periarticular width cut off value for the assessment of TMJ active inflammation used in two previous studies by Weiss, Arabshahi  and M\u00fcllFurther, the lower US LPAS mean value observed in our study could be influenced by the mandibular condylar cortical level where the LPAS measurements were performed. To reduce the changes in width due to disc displacement, Kirkhus, Gunderson  measuredThe Spearman correlation coefficient showed a positive significant correlation between MRI and US measurements of LPAS. A similar correlation was found by Kirkhus, Gunderson  between According to the literature, the present study findings suggest that US could be considered a clinically acceptable imaging modality to evaluate the TMJ involvement in patients affected by JIA , 24, 25.In children without clinical signs and symptoms of TMJ involvement or in the presence of unclear cases after the evaluation of the five clinical variables used by Stoustrup, Herlin  as indicThe strength of this study is that it is a prospective study with a control group. The JIA patients undertook the two imaging techniques (US and CE-MRI) at a maximum of 1\u00a0week apart, the same US and MRI scanners were used for all the patients and the same expert radiologist evaluated both the MRI and US images.Our study presents some limitations. First, the sample size was small and patients were referred by the Pediatrics Clinic of only one hospital. Thus, some categories of JIA were not present, and oligoarthritic and polyarthritis were more frequent than in epidemiological studies , respectCurrently, US cannot replace MRI but it could be used as a supplementary imaging method to assess temporomandibular joint involvement in JIA patients; especially in those cases when MRI is not available or children are not cooperative, and for the initial screening of TMJ involvement or the follow-up of the disease progression to avoid repeated MRI checks and related costs."
\ No newline at end of file