Membrane Tube Research Articles

Separation and Recycling of Concentrated Heavy Metal Wastewater by Tube Membrane Distillation Integrated with Crystallization.

Tube membrane distillation (MD) integrated with a crystallization method is used in this study for the concurrent productions of pure water and salt crystals from concentrated single and mixed system solutions. The effects of concentrated Zn2+ and Ni2+ on performance in terms of membrane flux, permeate conductivity, crystal recovery rates, and crystal grades are investigated. Preferred crystallization and co-crystallization determinations were performed for mixed solutions. The results revealed that membrane fluxes remained at 2.61 kg·m−2·h−1 and showed a sharp decline until the saturation increased to 1.38. Water yield conductivity was below 10 μs·cm−1. High concentrated zinc and nickel did not have a particular effect on the rejection of the membrane process. For the mixed solutions, membrane flux showed a sharp decrease due to the high saturation, while the conductivity of permeate remained below 10 μs·cm−1 during the whole process. Co-crystallization has been proven to be a better method due to the existence of the SO42− common-ion effect. Membrane fouling studies have suggested that the membrane has excellent resistance to fouling from highly concentrated solutions. The MD integrated with crystallization proves to be a promising technology for treating highly concentrated heavy metal solutions.

Cytonemes Versus Neutrophil Extracellular Traps in the Fight of Neutrophils with Microbes.

Neutrophils can phagocytose microorganisms and destroy them intracellularly using special bactericides located in intracellular granules. Recent evidence suggests that neutrophils can catch and kill pathogens extracellularly using the same bactericidal agents. For this, live neutrophils create a cytoneme network, and dead neutrophils provide chromatin and proteins to form neutrophil extracellular traps (NETs). Cytonemes are filamentous tubulovesicular secretory protrusions of living neutrophils with intact nuclei. Granular bactericides are localized in membrane vesicles and tubules of which cytonemes are composed. NETs are strands of decondensed DNA associated with histones released by died neutrophils. In NETs, bactericidal neutrophilic agents are adsorbed onto DNA strands and are not covered with a membrane. Cytonemes and NETs occupy different places in protecting the body against infections. Cytonemes can develop within a few minutes at the site of infection through the action of nitric oxide or actin-depolymerizing alkaloids of invading microbes. The formation of NET in vitro occurs due to chromatin decondensation resulting from prolonged activation of neutrophils with PMA (phorbol 12-myristate 13-acetate) or other stimuli, or in vivo due to citrullination of histones with peptidylarginine deiminase 4. In addition to antibacterial activity, cytonemes are involved in cell adhesion and communications. NETs play a role in autoimmunity and thrombosis.

Structures and mechanical behaviors of soft nanotubes confining adhesive single or multiple elastic nanoparticles

The mechanical interaction between soft membrane nanotubes and confined nanoparticles plays essential roles in numerous cellular activities such as inter- and intracellular transport and signaling, membrane tubulation and pearling transformation driven by adsorbed nanocomponents, and cooperative cell uptake. The aim of this work is to theoretically analyze how structures and mechanical behaviors of particle-in-tube complexes are regulated by the particle elasticity, covering the cases of a single particle and multiple particles of spatial periodicity. Depending on the interplay of membrane tension, adhesion energy, nanoparticle size, and the rigidity ratio between the nanoparticles and soft tubes, characteristic interaction states in terms of wrapping degrees of the confined particles have been identified, and the corresponding wrapping phase diagrams are determined. As the particles become softer, the adhesion energy required for the partial-wrapping state becomes lower but that for full-wrapping is larger, and a rich variety of characteristic system configurations are observed including the bulged or undulated cylinders, necklaces of spheres or prolates, and sausage-string shapes. Moreover, two fundamental modes of interaction between multiple nanoparticles and the soft nanotube are revealed, the cooperative wrapping and individual wrapping. Based on perturbation analysis at small tube deformation and configurational assumptions of catenoid, torus, and one-sheeted hyperboloid at more general tube deformation, we have also obtained analytical solutions on the wrapping of rigid spherical nanoparticles in soft tubes. Comparison between wrapping of elastic particles by a flat membrane and by a soft membrane tube is discussed. This study enriches our knowledge on the diversity of membrane tubules in morphologies and structures.

Radial aggregation of proteins prevails over axial aggregation on membrane tubes.

Tubular membrane structures are abundant in living cells and participate in various cell activities with the help of specific proteins. This complicated protein-membrane interaction raises a largely unclear question of how cells create, maintain and eliminate membrane nanotubes with a variety of proteins involved. To simplify the question and to give a common mechanism, we simply model membrane proteins as various solid nanoparticles (NPs) of different shapes, instead of considering the detailed structure of different proteins. With this minimal model of membrane proteins, we use molecular simulations to study the common features for protein self-assembly on membrane tubes. Both molecular simulations and energy analysis reveal that on tubular membrane surfaces, the radial aggregation structure of spherical NPs prevails over axial aggregation. We demonstrate that anisotropic deformation of membrane tubes by NP adhesion leads to a direction-dependent (effective) inter-NP attraction, which controls the direction of NP assembly. Moreover, this radial aggregation morphology seems to be independent of the shape of NPs, except for NPs with a length comparable to the tube diameter. This observation indicates that proteins with strong adhesion to a membrane tube tend to form ring-like aggregates, and this finding offers an insight into how proteins play their roles in generating, maintaining and breaking tubular membrane structures.

FIB-SEM-based analysis of Borrelia intracellular processing by human macrophages.

Borrelia burgdorferi is the causative agent of Lyme disease, a multisystemic disorder affecting primarily skin, joints and nervous system. Successful internalization and intracellular processing of borreliae by immune cells, like macrophages, is decisive for the outcome of a respective infection. Here, we use, for the first time, focused ion beam scanning electron microscopy tomography (FIB-SEM tomography) to visualize the interaction of borreliae with primary human macrophages with high resolution. We report that interaction between macrophages and the elongated and highly motile borreliae can lead to formation of membrane tunnels that extend deeper into the host cytoplasm than the actual phagosome, most probably as a result of partial extrication of captured borreliae. We also show that membrane tubulation at borreliae-containing phagosomes, a process suggested earlier as a mechanism leading to phagosome compaction but hard to visualize in live-cell imaging, is apparently a frequent phenomenon. Finally, we demonstrate that the endoplasmic reticulum (ER) forms multiple STIM1-positive contact sites with both membrane tunnels and phagosome tubulations, confirming the important role of the ER during uptake and intracellular processing of borreliae.

Dynamics of passive and active membrane tubes.

Utilising Onsager's variational formulation, we derive dynamical equations for the relaxation of a fluid membrane tube in the limit of small deformation, allowing for a contrast of solvent viscosity across the membrane and variations in surface tension due to membrane incompressibility. We compute the relaxation rates, recovering known results in the case of purely axis-symmetric perturbations and making new predictions for higher order (azimuthal) m-modes. We analyse the long and short wavelength limits of these modes by making use of various asymptotic arguments. We incorporate stochastic terms to our dynamical equations suitable to describe both passive thermal forces and non-equilibrium active forces. We derive expressions for the fluctuation amplitudes, an effective temperature associated with active fluctuations, and the power spectral density for both the thermal and active fluctuations. We discuss an experimental assay that might enable measurement of these fluctuations to infer the properties of the active noise. Finally we discuss our results in the context of active membranes more generally and give an overview of some open questions in the field.

In-Office Tympanostomy Tube Placement Using Iontophoresis and Automated Tube Delivery Systems.

Objectives(1) To evaluate safety, tolerability, and technical success of lidocaine iontophoresis and a tympanostomy tube placement system for adults in an office setting and (2) to meet regulatory evidence requirements for new drugs and devices.Study DesignProspective, multicenter, single arm.SettingPatients were recruited in 8 community-based practices in the United States between June and September 2017.Subjects and MethodsThis study evaluated tympanic membrane anesthesia and tube placement in 30 adults. Anesthesia was achieved via iontophoresis of a lidocaine/epinephrine solution. Tube placement was conducted using an integrated myringotomy and tube delivery system. Tolerability of tube placement was measured using a patient-reported visual analog scale from 0 mm (no pain) to 100 mm (worst possible pain). Mean pain score was compared to a performance goal of 45 mm, where statistical superiority represents mild pain or less. Technical success and safety through 3 weeks postprocedure were evaluated.ResultsTwenty-nine (29/30, 96.7%) patients had tube(s) successfully placed in all indicated ears. One patient demonstrated inadequate tympanic membrane anesthesia, and no tube placement was attempted. The mean (SD) pain score of 9.4 (15.7) mm was statistically superior to the performance goal. There were no serious adverse events. Seven nonserious events were related to device, procedure, or drug: inadequate anesthesia (1), vertigo (1), and dizziness (1) at the time of procedure and ear discomfort (1), tube occlusion (2), and medial tube migration (1) postprocedure.ConclusionLidocaine iontophoresis provides acceptable tympanic membrane anesthesia for safe, tolerable, and successful in-office tube placement using an integrated myringotomy and tube delivery system.

Reconstruction of Average Subtracted Tubular Regions (RASTR) enables structure determination of tubular filaments by cryo-EM

As the field of electron microscopy advances, the increasing complexity of samples being produced demand more involved processing methods. In this study, we have developed a new processing method for generating 3D reconstructions of tubular structures. Tubular biomolecules are common throughout many cellular processes and are appealing targets for biophysical research. Processing of tubules with helical symmetry is relatively straightforward for electron microscopy if the helical parameters are known, but tubular structures that deviate from helical symmetry (asymmetrical components, local but no global order, etc) present myriad issues. Here we present a new processing technique called Reconstruction of Average Subtracted Tubular Regions (RASTR), which was developed to reconstruct tubular structures without applying symmetry. We explain the RASTR approach and quantify its performance using three examples: a simulated symmetrical tubular filament, a symmetrical tubular filament from cryo-EM data, and a membrane tubule coated with locally ordered but not globally ordered proteins.

Regulation of caveolae through cholesterol-depletion-dependent tubulation mediated by PACSIN2.

The membrane-shaping ability of PACSIN2 (also known as syndapin II), which is mediated by its F-BAR domain, has been shown to be essential for caveolar morphogenesis, presumably through the shaping of the caveolar neck. Caveolar membranes contain abundant cholesterol. However, the role of cholesterol in PACSIN2-mediated membrane deformation remains unclear. Here, we show that the binding of PACSIN2 to the membrane can be negatively regulated by cholesterol. We prepared reconstituted membranes based on the lipid composition of caveolae. The reconstituted membrane with cholesterol had a weaker affinity for the F-BAR domain of PACSIN2 than a membrane without cholesterol. Consistent with this, upon depletion of cholesterol from the plasma membrane, PACSIN2 localized at tubules that had caveolin-1 at their tips, suggesting that cholesterol inhibits membrane tubulation mediated by PACSIN2. The tubules induced by PACSIN2 could be representative of an intermediate of caveolae endocytosis. Consistent with this, the removal of caveolae from the plasma membrane upon cholesterol depletion was diminished in the PACSIN2-deficient cells. These data suggest that PACSIN2-mediated caveolae internalization is dependent on the amount of cholesterol, providing a mechanism for cholesterol-dependent regulation of caveolae.This article has an associated First Person interview with the first author of the paper.

Applications of Membrane Hydrocyclone Recovery of Silicon Carbide Powder

This work investigated the possibility of using membrane hydrocyclone as a separator to recover the silicon carbide powder. The weight percentage of silicon carbide powder is 62.9%. Classifying these powders by a 2.5 cm-diameter hydrocyclone that equips membrane tube in the center position, overflow and underflow are obtained. The results of the primary separation show that the overflow and underflow are about half of the proportion of solid, but silicon carbide powder content in the overflow is higher. Therefore, it is necessary to consider the recovery of silicon carbide powder contained in the overflow.

Cultivation of Leptospira interrogans Within Rat Peritoneal Dialysis Membrane Chambers.

In order to sustain its zoonotic lifecycle, leptospires must adapt to growth within the host milieu. Signals encountered within the mammal also trigger regulatory programs required by Leptospira for the expression of virulence-related gene products. The complex transcriptional, antigenic, and physiological changes leptospires undergo within the mammal are collectively referred to as "host adaptation." In this chapter, we describe the procedures for the generation of host-adapted Leptospira spp. by cultivation within dialysis membrane chambers (DMCs) implanted in rat peritoneal cavities. In this model, Leptospira spp. diluted in EMJH medium are sequestered within sterile dialysis membrane tubing closed at both ends. The chamber then is surgically implanted within the peritoneal cavity of a rat and incubated for 7-10days. During this period, leptospires are exposed to many, if not all, of the physiological and nutritional cues required for host adaptation while at the same time protected from clearance by host innate and adaptive immune defenses.

Dynamics and spectral stability of soliton-like structures in fluid-filled membrane tubes

В обзоре излагаются результаты об устойчивости уединенных волн возвышения в осесимметричных упругих мембранных трубках, заполненных жидкостью. Материал упругой трубки характеризуется упругим потенциалом (упругой энергией), нелинейно зависящим от главных деформаций и описывающим податливые упругие среды. В обзоре используется простая модель невязкой несжимаемой жидкости, которая тем не менее позволяет отследить основные закономерности динамики уединенных волн. К одной из таких закономерностей относится спектральная устойчивость (линейная устойчивость по форме) этих волн. Формулируются основные уравнения системы "осесимметричная трубка-идеальная жидкость", причем в уравнениях для жидкости производится осреднение по поперечному сечению трубки, т. е. рассматривается квазиодномерное течение с волнами, длина которых существенно превосходит радиус трубки. Изучение спектральной устойчивости относительно осесимметричных возмущений производится при помощи построения функции Эванса для линеаризованной вокруг решения типа уединенной волны системы основных уравнений. Функция Эванса зависит только от спектрального параметра $\eta$ и аналитична в правой комплексной полуплоскости $\Omega^+$, а ее нули в $\Omega^+$ совпадают с неустойчивыми собственными значениями. Рассматриваются задачи об устойчивости неподвижных уединенных волн в случае отсутствия жидкости внутри трубки (случай постоянного внутреннего давления), в том числе и для локальной неоднородности (утончения) стенки трубки, в случаях неподвижной жидкости, заполняющей трубку (случай нулевого среднего течения), и движущейся жидкости, а также задачи об устойчивости бегущих уединенных волн, распространяющихся вдоль трубки с ненулевой скоростью. Библиография: 83 названия.

Construction of flexible enzymatic electrode based on gradient hollow fiber membrane and multi-wall carbon tubes meshes

In this study, we developed a convenient way to construct a flexible enzymatic electrode with excellent stability and electrochemical performance for implanted glucose monitoring. The electrode was constructed through the co-immobilization of the glucose oxidase micro-particles (GOD MPs) and multi-wall carbon nanotubes (CNT) on the inner surface of a gradient-structured hollow fiber membrane (GHM), where CNT improved the electron transport efficiency and GHM controlled the transfer of substances and interferences. GOD MPs showed higher stability under various operation conditions than the free enzymes due to the MnCO3 template method, which enabled the biosensor to remain relative sensitivity at >86% over 9 days. The GOD MPs biosensor also showed high selectivity, reproducibility, and linear sensing range from 0 mM to 24 mM (R2 = 0.9993) with a current sensitivity of 25 nA/mM. The combination of porous-structured membrane and the flexible CNT meshes ensures the electrical connections and sensing accuracy of the biosensor under the deformation status. In-vivo experiments showed reliable current responses to variations in blood glucose concentrations that were consistent with tail blood test results. This co-immobilization of enzyme micro-particles in the 3D porous structure method developed a bio-composite platform technology towards the applications in flexible sensing and implantable medical devices.

Cells Control BIN1-Mediated Membrane Tubulation by Altering the Membrane Charge

The Bridging integrator 1 (BIN1)/Amphiphysin/Rvs (BAR) protein family is an essential part of the cell's machinery to bend membranes. BIN1 is a muscle-enriched BAR protein with an established role in muscle development and skeletal myopathies. Here, we demonstrate that BIN1, on its own, is able to form complex interconnected tubular systems in vitro, reminiscent of t-tubule system in muscle cells. We further describe how BIN1's electrostatic interactions regulate membrane bending: the ratio of negatively charged lipids in the bilayer altered membrane bending and binding properties of BIN1 and so did the manipulation of BIN1's surface charge. We show that the electrostatically mediated BIN1 membrane binding depended on the membrane curvature—it was less affected in liposomes with high curvature. Curiously, BIN1 membrane binding and bending was diminished in cells where the membrane's charge was experimentally reduced. Membrane bending was also reduced in BIN1 mutants where negative or positive charges in the BAR domain have been eliminated. This phenotype, characteristic of BIN1 mutants linked to myopathies, was rescued when the membrane charge was made more negative. The latter findings also show that cells can control tubulation at their membranes by simply altering the membrane charge and through it, the recruitment of BAR proteins and their interaction partners (e.g. dynamin).

CFD simulation of flow field and resistance in a 19-core tandem ceramic membrane module

Abstract CFD simulation of the permeation process of a 19-core tandem ceramic membrane module was established to investigate flow field and resistance and its change in permeate flux to the membrane element position and the channel of each membrane element. The results show that when the volume flow rate changes from 26 m3·h−1 to 89 m3·h−1, the resistance of each part of the membrane module increases gradually. The increase in resistance loss in the membrane element is faster than the plates and the bell mouths. In a single ceramic membrane module, the maximum difference in flow rate of each membrane tube is 7.23%. In a single membrane tube, the outer ring channels 3–5, 3–6, 3–7, 3–8 are relatively slow. The maximum mass flow deviation from the mean is 2.7%. This work helps to clarify the flow mechanism within the modules, optimize the structure of the equipment and provide a reliable basis for the improvement of industrial ceramic membrane modules.

Expression, Purification, and Liposome Binding of Budding Yeast SNX-BAR Heterodimers.

SNX-BAR proteins are an evolutionarily conserved class of membrane remodeling proteins that play key roles in sorting and trafficking of protein and lipids during endocytosis, sorting within the endosomal system, and autophagy. Central to SNX-BAR protein function is the ability to form homodimers or heterodimers that bind membranes using highly conserved phox-homology (PX) and BAR (Bin/Amphiphysin/Rvs) domains. In addition, oligomerization of SNX-BAR dimers on membranes can elicit the formation of membrane tubules and vesicles and this activity is thought to reflect their functions as coat proteins for endosome-derived transport carriers. Researchers have long utilized in vitro binding studies using recombinant SNX-BAR proteins on synthetic liposomes or giant unilamellar vesicles (GUVs) to reveal the precise makeup of lipids needed to drive membrane remodeling, thus revealing their mechanism of action. However, due to technical challenges with dual expression systems, toxicity of SNX-BAR protein expression in bacteria, and poor solubility of individual SNX-BAR proteins, most studies to date have examined SNX-BAR homodimers, including non-physiological dimers that form during expression in bacteria. Recently, we have optimized a protocol to overcome the major shortcomings of a typical bacterial expression system. Using this workflow, we demonstrate how to successfully express and purify large amounts of SNX-BAR heterodimers and how to reconstitute them on synthetic liposomes for binding and tubulation assays.

Morphofunctional changes in the heart and lung tissues of dogs for isoniazid poisoning

The article presents the results of macroscopic, histological and histochemical studies of the heart and lungs of dogs for isoniazid poisoning. A pathoanatomical section of 19 carcasses of dogs of different ages and breeds was performed; heart and lung fragments were selected for morphological examination. Heart and lung samples were fixed in 10 % aqueous formalin neutral solution, Carnua and Buen fluids. Dehydration was carried out in ethanol of increasing concentration and embedded in paraffin. The sections were made on a MC-2 microtome 7 μm thick, stained with hematoxylin and eosin, using the McManus and Brasche technique. Made histopreparations were examined under a Leica DM 2500 light microscope (Switzerland), photo-fixation was performed with a Leica DFC450C using Leica Application Suite Version 4.4 software. The results of morphohistochemical studies have shown that in acute poisoning of dogs with isoniazid in the heart prevailed acute disorders of the vascular circulation, manifested by a sharp expansion and destruction of the walls of the venous vessels, plasmorrhagia, microtromb of the capillaries and spasm of the arterioles, which led to the development of swelling, caused acute decrease of protein synthesation function of muscle fibers and dystrophic-necrobiotic changes of cardiomyocytes. It was noted, in the lungs of dogs on the background of acute congestive hyperemia, diapedesis of erythrocytes and accumulation of transudate in the lumen of the alveoli, foci of emphysema and bronchiectasis with rupture of the epithelial layer of the mucous membrane, elastic skeleton and muscular tubules of bronchial tubules that indicated the violation of gas exchange and the development of anoxemia. It is known that spontaneous isoniazid poisoning in dogs leads to an acute course and often fatal consequences. In the comprehensive study of the effects of chemicals on the body, and in particular isoniazid on the body of dogs, toxicological and pathomorphological studies are of great importance. The use of morphological and histochemical methods will reveal some of the mechanisms of intoxication.

Dynamic Growth and Shrinkage of the Salmonella-Containing Vacuole Determines the Intracellular Pathogen Niche

SummarySalmonella is a human and animal pathogen that causes gastro-enteric diseases. The key to Salmonella infection is its entry into intestinal epithelial cells, where the bacterium resides within a Salmonella-containing vacuole (SCV). Salmonella entry also induces the formation of empty macropinosomes, distinct from the SCV, in the vicinity of the entering bacteria. A few minutes after its formation, the SCV increases in size through fusions with the surrounding macropinosomes. Salmonella also induces membrane tubules that emanate from the SCV and lead to SCV shrinkage. Here, we show that these antipodal events are utilized by Salmonella to either establish a vacuolar niche or to be released into the cytosol by SCV rupture. We identify the molecular machinery underlying dynamic SCV growth and shrinkage. In particular, the SNARE proteins SNAP25 and STX4 participate in SCV inflation by fusion with macropinosomes. Thus, host compartment size control emerges as a pathogen strategy for intracellular niche regulation.

The genesis of agates and amethyst geodes

AbstractPractically all aspects of agate genesis generate debate. The time is ripe to clarify the most important enigmas concerning the environments of formation of agates and the related famous amethyst geodes of Brazil and Uruguay. Agates form over a wide range of temperatures, from those of basaltic and andesitic melts (about 1100 °C) down to about 50 °C, and at rather low pressures. Their formation in liquid mafic magmas is indicated by a correlation between (1) the orientation of amygdules and the inclination of onyx banding in them and (2) the attitude of amygdules in the lava flow layers. The correlation arises because lava moves at a different rate close to and far from the upper and lower rims of a flow. The alkaline supercritical fluid fills gas vesicles in lavas and dissolves silica, mainly, from ambient lava or rock to produce a silica sol. If the pressure on the fluid causes percolation of water from amygdules, the sol coagulates on the walls of the vesicle to form a concentric lining. If the pressure in amygdules falls below the maximum osmotic pressure of a sol (about 0.1 MPa for a silica sol), percolation of fluid stops, and coagulation leads to the formation of horizontal onyx banding. Multiple repetitions of precipitation of various gel layers can be caused by overlapping fresh flows upon the cooling older agate-bearing lava flow. In a submarine setting, phase separation of the fluid and the formation of a film of gel between vapor (or diluted solution) and brine stimulate the osmotic processes, which result in growth of hollow membrane tubes and branching moss-like arrays at the bottom of amygdules. Some agates exhibit numerous channels as a result of repeated extrusion of fluid or gel from inner zones to the periphery of amygdules that were compressed under the burden of new flows. Previously, such channels were interpreted to be feeding channels for silica supply in amygdules. Periodic compression of amygdules after percolation of fluid from them requires no additional supply of silica because the volume of the amygdules is reduced in proportion to the loss of fluid. The concentric and horizontal banding and mossy textures of agates from the lithophysae of felsic volcanic rocks were created during active volcanism as well. The agates from dissolution-induced cavities in carbonate rocks and the famous amethyst druses of Brazil and Uruguay formed at the moderate temperatures associated with low-grade burial metamorphism, as indicated by the lack of moss textures and onyx banding.

SEPTUM VAGINA TRANSVERSA

The vagina is a membranous muscular tube that connects the vulva and uterus. Congenital or congenital abnormalities in the form of complete or partial absence of the vagina (vaginal agenesis). Patients who experience vaginal agenesis have less frequency, namely 1 in 4000 births, 1 in 4000 to 10,000 births (ACOG). Meanwhile in Dr. Cipto Mangunkusumo Jakarta from 1995 to 1999, an average of 10-12 cases per year. It has been reported the case of a female patient aged 14 years who entered the Gynecology Ward Dr. M. Djamil Padang on January 25, 2014 at 11.00 WIB with a delivery from the RSMJ gyn clinic with a diagnosis of transverse vaginal septum + hematometra + hematotrachelos + hematokolpos planned for a septal incision. After performing the operative action, ± 500 cc of blackish brown liquid was successfully removed.Keywords: Agenesis Vagina, TVS, Tranverse Septum Vagina, Case Report