Analysis of Cell Membrane Characteristics of In Vitro-Selected

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Elabscience

（本试剂盒仅供体外研究使用，不用于临床诊断！）Elabscience®过氧化氢（H2O2）比色法测试盒Hydrogen Peroxide (H2O2) Colorimetric Assay Kit产品货号：E-BC-K102-M产品规格：48T(32 samples)/96T(80 samples)检测仪器：酶标仪(402-407 nm)使用前请仔细阅读说明书。

如果有任何问题，请通过以下方式联系我们：销售部电话************，************技术部电话131****6790具体保质期请见试剂盒外包装标签。

请在保质期内使用试剂盒。

联系时请提供产品批号(见试剂盒标签)，以便我们更高效地为您服务。

用途本试剂盒适用于检测血清、血浆、尿液、动植物组织及细胞样本中的H2O2含量。

检测原理H2O2与钼酸铵反应生成稳定的黄色络合物且在405 nm处有最大吸收，黄色络合物的颜色深浅与H2O2的浓度在一定范围内具有线性关系。

故可以通过比色计算出H2O2的含量。

本试剂盒检测组织和细胞样本时，需测定总蛋白浓度，推荐使用考马斯亮蓝法（货号：E-BC-K168-M）。

提供试剂和物品说明：试剂严格按上表中的保存条件保存，不同测试盒中的试剂不能混用。

对于体积较少的试剂，使用前请先离心，以免量取不到足够量的试剂。

所需自备物品仪器：酶标仪（402-407 nm，最适检测波长405 nm）、涡旋混匀仪、37℃恒温箱、微量移液器（1000 μL，200 μL，100 μL，10 μL）、离心机。

耗材：枪头（1000 μL，200 μL，10 μL）、EP管（2 mL、10 mL）。

试剂：双蒸水、生理盐水（0.9% NaCl）或PBS（0.01 M，pH 7.4）。

试剂准备①实验开始前将所有试剂平衡至室温。

②不同浓度标准品的稀释样本准备①样本处理血清血浆等液体样本：可直接测定。

组织样本：常规匀浆处理(生理盐水(0.9% NaCl溶液)或PBS(0.01 M，pH 7.4)。

Amicon 15ml超滤离心管使用说明书

formation in this document is subject to change without notice and should not be construed as a commitment by Millipore Corporation or an affiliated corporation. Neither Millipore Corporation nor any of its affiliated corporations assumes responsibility for any errors that may appear in this document. This manual is believed to be complete and accurate at the time of publication. In no event shall Millipore Corporation be liable for incidental or consequential damages in connection with or arising from the use of this manual.
User Guide
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Amicon Ultra-15 10K device for in vitro diagnostic use
Amicon Ultra-15 3K, 30K, 50K, and 100K devices for research use only; not for use in diagnostic procedures
Contents
Introduction..........................................................................................................................................................1 Applications..........................................................................................................................................................2 Materials Supplied................................................................................................................................................3 Required Equipment............................................................................................................................................4 Suitability..............................................................................................................................................................4 Device Storage.....................................................................................................................................................4 Specifications.......................................................................................................................................................5 Chemical Compatibility.........................................................................................................................................7 How to Use Amicon Ultra-15 Centrifugal Filter Devices........................................................................................9 How to Quantitate Recoveries............................................................................................................................10 Performance - Protein Concentration.................................................................................................................13

《新型冠状病毒感染合并急性肾损伤诊治专家共识》解读

SARS-CoV-2 感染主要引起呼吸、心血管、消化、泌尿、神经等多系统器官疾病，其临床表现与受累系统相一致。本指南重点介绍了肾脏受累后相关临床表现及实验室检查等，但缺乏对肾脏病理的阐述，现就肾脏病理进行补充：COVID-19 病毒并发急性肾损伤的患者，其肾脏病理学的主要表现包括急性肾小管坏死、急性间质性肾炎、足细胞病 / 塌陷型局灶节段性肾小球硬化、微血管病等。 [17] 4 诊断
[2] TANG Y, XIN Y, DENG F. Prevention and management of COVID-19 in hemodialysis centers[J]. Am J Manag Care, 2020, 26(8): e237-e238.
[3] KLIGER A, COZZOLINO M, JHA V, et al. Managing the COVID-19 pandemic: international comparisons in dialysis patients[J]. Kidney Int, 2020, 98(1): 12-16.
[6] CHAWKI S, BUCHARD A, SAKHI H, et al. Treatment impact on COVID-19 evolution in hemodialysis patients[J]. Kidney Int, 2020, 98(4): 1053-1054.
[7] MURT A, ALTIPARMAK M R. Convalescent COVID-19 patients on hemodialysis: when should we end isolation?[J]. Nephron, 2020, 144(7): 343-344. 收稿日期：2021-03-25；修回日期：2021-04-22 （本文编辑：王丽）

细胞的基本特性

BASIC PROPERTIES OF CELLS Just as plants and animals are alive, so too are cells. Life, in fact, is the most basic property of cells, and cells are the smallest units to exhibit this property. Unlike the parts of a cell, which simply deteriorate if isolated, whole cells can be removed from a plant or animal and cultured in a laboratory where they will grow and reproduce for extended periods of time. If mistreated, they may die.Death can also be considered one of the most basic properties of life, because only a living entity faces this prospect. Remarkably, cells within the body generally die “by their own hand”—the victims of an internal program that causes cells that are no longer needed or cells that pose a risk of becoming cancerous to eliminate themselves.The first culture of human cells was begun by George and Martha Gey of Johns Hopkins University in 1951. The cells were obtained from a malignant tumor and named HeLa cells after the donor, Henrietta Lacks. HeLa cells—descended by cell division from this first cell sample—are still being grown in laboratories around the world today. Because they are so much simpler to study than cells situated within the body, cells grown in vitro (i.e., in culture, outside the body) have become an essential tool of cell and molecular biologists. In fact, much of the information that will be discussed in this book has been obtained using cells grown in laboratory cultures.We will begin our exploration of cells by examining a few of their most fundamental properties.Cells Are Highly Complex and OrganizedComplexity is a property that is evident when encountered, but difficult to describe. For thepresent, we can think of complexity in terms of order and consistency.The more complex a structure, the greater the number of parts that must be in their proper place, the less tolerance of errors in the nature and interactions of the parts, and the more regulation or control that must be exerted to maintain the system.Cellular activities can be remarkably precise. DNA duplication, for example, occurs with an error rate of less than one mistake every ten million nucleotides incorporated—and most of these are quickly corrected by an elaborate repair mechanism that recognizes the defect.During the course of this book, we will have occasion to consider the complexity of life at several different levels. We will discuss the organization of atoms into small-sized molecules; the organization of these molecules into giant polymers; and the organization of different types of polymeric molecules into complexes, which in turn are organized into subcellular organelles and finally into cells. As will be apparent, there is a great deal of consistency at every level.Each type of cell has a consistent appearance when viewed under a high-powered electron microscope; that is, its organelles have aparticular shape and location, from one individual of a species to another. Similarly, each type of organelle has a consistent composition of macromolecules, which are arranged in a predictable pattern.Consider the cells lining your intestine that are responsible for removing nutrients from your digestive tract.The epithelial cells that line the intestine are tightly connected to each other like bricks in a wall. The apical ends of these cells, which face the intestinal channel, have long processes (microvilli) that facilitate absorption of nutrients. The microvilli are able to project outward from the apical cell surface because they contain an internal skeleton made of filaments, which in turn are composed of protein (actin) monomers polymerized in a characteristic array. At their basal ends, intestinal cells have large numbers of mitochondria that provide the energy required to fuel various membrane transport processes. Each mitochondrion is composed of a defined pattern of internal membranes, which in turn are composed of a consistent array of proteins, including an electrically powered ATP-synthesizing machine that projects from the inner membrane like a ball on a stick.Fortunately for cell and molecular biologists, evolution has moved rather slowly at the levels of biological organizasigtion with which they are concerned. Whereas a human and a cat, for example, have very different anatomical features, the cells that make up their tissues, and the organelles that make up their cells, are very similar. The actin filament portrayed in Figure 1.3, Inset 3, and the ATP-synthesizing enzyme of Inset 6 are virtually identical to similar structures found in such diverse organisms as humans, snails, yeast, and redwood trees. Information obtained by studying cells from one type of organism often has direct application to other forms of life. Many of the most basic processes, such as the synthesis of proteins, the conservation of chemical energy, or the construction of a membrane, are remarkably similar in all living organisms.Cells Possess a Genetic Program and the Means to Use ItOrganisms are built according to information encoded in a collection of genes. The human genetic program contains enough information, ifconverted to words, to fill millions of pages of text. Remarkably, this vast amount of information is packaged into a set of chromosomes that occupies the space of a cell nucleus—hundreds of times smaller than the dot on this i.Genes are more than storage lockers for information: they constitute the blueprints for constructing cellular structures, the directions for running cellular activities, and the program for making more of themselves. The molecular structure of genes allows for changes in genetic information (mutations) that lead to variation among individuals, which forms the basis of biological evolution. Discovering the mechanisms by which cells use their genetic information has been one of the greatest achievements of science in recent decades.Cells Are Capable of Producing More of ThemselvesJust as individual organisms are generated by reproduction, so too are individual cells. Cellsreproduce by division, a process in which the contents of a “mother” cell are distributed into two “daughter” cells. Prior to division, the genetic material is faithfully duplicated, and each daughter cell receives a complete and equal share of genetic information. In most cases, the two daughter cells have approximately equal volume. In some cases, however, as occurs when a human oocyte undergoes division, one of the cells can retain nearly all of the cytoplasm, even though it receives only half of the genetic materialCells Acquire and Utilize EnergyEvery biological process requires the input of energy. Virtually all of the energy utilized by life on the Earth’s surface arrives in the form of electromagnetic radiation from the sun. The energy of light is trapped by light-absorbing pigments present in the membranes of photosynthetic cells.Light energy is converted by photosynthesisinto chemical energy that is stored in energy-rich carbohydrates, such as sucrose or starch. For most animal cells, energy arrives prepackaged, of-ten in the form of the sugar glucose. In humans, glucose is released by the liver into the blood where it circulates through the body delivering chemical energy to all the cells. Once in a cell, the glucose is disassembled in such a way that its energy content can be stored in a readily available form (usually as ATP) that is later put to use in running all of the cell’s myriad energy-requiring activities. Cells expend an enormous amount of energy simply breaking down and rebuilding the macromolecules and organelles of which they are made. This continual “turnover,” as it is called, maintains the integrity of cell components in the face of inevitable wear and tear and enables the cell to respond rapidly to changing conditions.Cells Carry Out a Variety of Chemical ReactionsCells function like miniaturized chemical plants.Even the simplest bacterial cell is capable of hundreds of different chemical transformations, none of which occurs at any significant rate in the inanimate world. Virtually all chemical changes that take place in cells require enzymes—molecules that greatly increase the rate at which a chemical reaction occurs. The sum total of the chemical reactions in a cell represents that cell’s metabolism.Cells Engage in Mechanical ActivitiesCells are sites of bustling activity. Materials are transported from place to place, structures are assembled and then rapidly disassembled, and, in many cases, the entire cell moves itself from one site to another. These types of activities are based on dynamic, mechanical changes within cells, many of which are initiated by changes in the shape of “motor” proteins. Motor proteins are just one of many types of molecular “machines” employed by cells to carry out mechanical activities.Cells Are Able to Respond to StimuliSome cells respond to stimuli in obvious ways;a single-celled protist, for example, moves away from an object in its path or moves toward a source of nutrients. Cells within a multicellular plant or animal respond to stimuli less obviously. Most cells are covered with receptors that interact with substances in the environment in highly specific ways. Cells possess receptors to hormones, growth factors, and extracellular materials, as well as to substances on the surfaces of other cells. A cell’s receptors provide pathways through which external agents can evoke specific responses in target cells. Cells may respond to specific stimuli by altering their metabolic activities, moving from one place to another, or even committing suicide.Cells Are Capable of Self-RegulationIn addition to requiring energy, maintaining a complex, ordered state requires constant regulation. The importance of a cell’s regulatorymechanisms becomes most evident when they break down. For example, failure of a cell to correct a mistake when it duplicates its DNA may result in a debilitating mutation, or a breakdown in a cell’s growth-control safeguards can transform the cell into a cancer cell with the capability of destroying the entire organism. We are gradually learning how a cell controls its activities, but much more is left to discover.Consider the following experiment conducted in 1891 by Hans Driesch, a German embryologist.Driesch found that he could completely separate the first two or four cells of a sea urchin embryo and each of the isolated cells would proceed to develop into a normal embryo (Figure 1.6). How can a cell that is normally destined to form only part of an embryo regulate its own activities and form an entire embryo? How does the isolated cell recognize the absence of its neighbors, and how does this recognition redirect the course of the cell’s development? How can apart of an embryo have a sense of the whole? We are not able to answer these questions much better today than we were more than a hundred years ago when the experiment was performed. Throughout this book we will be discussing processes that require a series of ordered steps, much like the assembly line line construction of an automobile in which workers add, remove, or make specific adjustments as the car moves along. In the cell, the information for product design resides in the nucleic acids, and the construction workers are primarily proteins. It is the presence of these two types of macromolecules that, more than any other factor, sets the chemistry of the cell apart from that of the nonliving world. In the cell, the workers must act without the benefit of conscious direction. Each step of a process must occur spontaneously in such a way that the next step is automatically triggered. In many ways, cells operate in a manner analogous to the orange-squeezing contraption discovered by “TheProfessor” and shown in Figure 1.7. Each type of cellular activity requires a unique set of highly complex molecular tools and machines—the products of eons of natural selection and biological evolution. A primary goal of biologists is to understand the molecular structure and role of each component involved in a particular activity, the means by which these components interact, and the mechanisms by which these interactions are regulated.Cells EvolveHow did cells arise? Of all the major questions posed by biologists, this question may be the least likely ever to be answered. It is presumed that cells evolved from some type of precellular life form, which in turn evolved from nonliving organic materials that were present in the primordial seas. Whereas the origin of cells is shrouded in near-total mystery, the evolution of cells can be studied by examining organisms that are alive today. If you were to observe the features of a bacterialcell living in the human intestinal tract (see Figure 1.18a) and a cell that is part of the lining of that tract (Figure 1.3), you would be struck by the differences between the two cells. Yet both have evolved from a common ancestral cell that lived more than three billion years ago. Those structures that are shared by these two distantly related cells, such as their similar plasma membrane and ribosomes, must have been present in the ancestral cell. We will examine some of the events that occurred during the evolution of cells in the Experimental Pathways at the end of the chapter. Keep in mind that evolution is not simply an event of the past, but aan ongoing process that continues to modify the properties of cells that will be present in organisms that have yet to appear.。

transwell_guide,细胞小室用法说明

Table of ContentsIntroduction . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 Selecting the Right T ranswell Membraneand Pore Size . . . . . . . . . . . . . . . . . . . . . . . . . .2 Selecting the Right T ranswell System . . . . . .4Using Transwell Permeable Supports . . . . . .5 Helpful Hints . . . . . . . . . . . . . . . . . . . . . . . . . .5 General Directions for Use . . . . . . . . . . . . . . .6Technical Assistance . . . . . . . . . . . . . . . . . . . . .7Ordering Information . . . . . . . . . . . . . . . . . . . .8 Polyester (PET) T ranswell Inserts . . . . . . . . .8 Collagen-Coated T ranswell-COL Inserts . . .8 Polycarbonate T ranswell Inserts . . . . . . . . . .9 Snapwell™Inserts . . . . . . . . . . . . . . . . . . . . . .9 HTS T ranswell-24 Systems . . . . . . . . . . . . . .10 HTS T ranswell-96 Systems . . . . . . . . . . . . . .11 6, 12, and 24 Well Cell Culture Plates . . . . .12IntroductionCell and tissue culture techniques are becoming increasingly important for basic and applied life science research. The development of new culture vessels and cell attachment substrates is currently being driven by the need to produce an environ-ment that resembles the in vivo state as closely as possible to enable the growth of specialized cell types. Consequently, using permeable supports with microporous membranes have become a standard method for culturing these cells. These permeable supports have allowed significant improvements in culturing polarized cells since these permeable supports permit cells to uptake and secrete molecules on both their basal and apical surfaces and thereby carry out metabolic activities in a more natural fashion.Membrane filters have been used as cell growth substrates since the transfilter metanephric induc-tion studies of Grobstein (Nature, 172:860-872; 1953). Adapted over the years to a variety of cell types and numerous applications, permeable sup-ports treated for cell growth are now recognized as providing significant advantages over solid, impermeable cell growth substrates. For epithelial and other cell types, the use of permeable supportsTranswell®Permeable SupportsSelection and Use GuideLifeSciencesin vitro allows cells to be grown and studied in a polarized state under more natural conditions. Cellular differentia-tion can also proceed to higher levels resulting in cells that morphologically |and functionally better represent their in vitro counterparts.Cellular functions such as transport, adsorp-tion and secretion can also be studied since cells grown on permeable supports provide convenient, independent access to apical and basolateral plasma mem-brane domains. The use of permeable support systems for cell culture has proven to be an invaluable tool in the cell biology laboratory. Selecting the Right Transwell ®Membrane and Pore Size T ranswell permeable supports are avail-able in three membrane materials: poly-carbonate (PC), polyester (PET), and collagen-coated polytetrafluoroethylene (PTFE). See T able 1 for additional infor-mation on these membrane characteristics. ◗Polyester T ranswell-Clear inserts fea-ture a microscopically transparent poly-ester membrane that is tissue culture treated for optimal cell attachment and growth. T ranswell-Clear inserts provide better cell visibility under phase con-trast microscopy and allow assessment of cell viability and monolayer forma-tion. ◗Polycarbonate T ranswell inserts are available in a variety of pore sizes rang-ing from 0.1 µm to 12.0 µm. Most are treated for optimal cell attachment.◗T ranswell-COL inserts have a transpar-ent (when wet), collagen-treated PTFE membrane that promotes cell attach-ment and spreading and allow cells to be visualized during culture. The T ranswell-COL membrane has an equimolar mixture of types I and III collagen derived from bovine placentas.Unlike traditional coating techniques that result in occluding film layers,Corning’s proprietary coating process results in a biologically stable collagen that covers every fibril of the filter matrix, thereby retaining the porosity of the membrane.Selecting Pore Sizes Selecting the correct pore size for experiments using T ranswell permeable supports is also very important. T able 2reviews common permeable support applications along with recommended pore sizes. The smallest pore size T ranswell membranes (0.1 µm) are pri-marily used in drug transport studies.Cell invasion, chemotaxis and motility studies are usually done in T ranswell membranes with 3.0 µm or larger pores.The ability of cells to migrate through pores of a membrane is dependent on the cell line used and the culture conditions,as well as the pore size. Cell migration will not occur with pores smaller than 3.0 µm. For critical experiments, Corning suggests experimenting with appropriate controls with a range of pore sizes to determine which size works best with your cell cultures and your specific application. As an alternative, followSEM of the surface of a0.4 µm pore polycarbonatemembraneSEM of a PTFE membraneshowing its pore structureThe polyester Transwell-Clearinserts in a 6 well plate showthe clarity of the membraneThese 12 mm diameterTranswell inserts have apolycarbonate membraneMD).recommendations in published scientific literature. For additional application and use information, please refer to the T ranswell ®Bibliography on the T echnical Information section of the Corning Life Sciences web site that lists over 800literature references using T ranswell permeable supports. Chemical Compatibility All of the T ranswell membranes are compatible with histological fixatives including methanol and formaldehyde.The polyester T ranswell membranes have the best overall chemical resistance.These membranes (but not the poly-styrene housings) are compatible with many alcohols, amines, esters, ethers,ketones, oils and some solvents including many halogenated hydrocarbons and DMSO, but are not recommended for use with strong acids and bases.Pore Density Of the three types of T ranswell mem-branes, only the collagen-coated PTFE membrane does not have a defined pore density because it is a tortuous path mem-brane. The two membranes with nominally defined pore densities are polycarbonate and polyester. The polyester T ranswell membranes do not have as high a pore density as the polycarbonate T ranswell membranes but have better optical clarity as a result. The nominal pore densities for Corning ®polycarbonate and poly-ester membranes are given in T able 3.Selecting the Right Transwell System T ranswell permeable support units come in three basic designs:◗T raditional T ranswell plate inserts that are used individually in 6, 12 and 24 well plates or 100 mm dishes;◗HTS T ranswell-24 and HTS T ranswell-96 insert systems that are mounted in special holders to allow for automation and ease of handling;◗Snapwell ™inserts for use in diffusion or Ussing chambers.More detailed information on each of these products is found below and in the ordering section.24.5 mm Transwell-COL insert being placed into a 6 well microplate.75 mm Transwell insert and 100 mm dish bottomTraditional Transwell ®Permeable Supports T ranswell inserts are available in four membrane diameters: 6.5 mm (24 well plate), 12 mm (12 well plate), 24 mm (6 well plate) and 75 mm (100 mm dish)formats. See T able 4 for cell growth areas provided by these sizes. Several membrane types and a large selection of pore sizes are available with each of these units. The patented self-centering design prevents medium from wicking between the sides of the insert and the well wall. The hanging design keeps the T ranswell membrane about a millimeter off the bottom of the well.This prevents co-cultured cell monolay-ers in the bottom of the well from being scratched or disturbed when the insert is moved. Windows or openings in the sides of the T ranswell insert allow access to the bottom compartment.HTS Transwell Systems The HTS T ranswell systems are arrays of either 24 or 96 individual T ranswell inserts connected by a rigid, robotics-friendly holder that enables all of the T ranswell-24 or T ranswell-96 inserts to be handled as a single unit. This makes HTS T ranswell systems ideal tools for running automated, high throughput drug transport (Caco-2 cells) or cell toxicity studies. The HTS T ranswell-96 culture system consists of 4 parts: a 96 well insert sup-port plate with a choice of either 1.0 µm pore polyester or 0.4 µm pore polycar-bonate membranes; a Reservoir Plate with a removable media stabilizer for feeding cultures; a 96 well Receiver Plate for use in assays; and two lids to minimize evaporation and protect against contami-nation. Each well insert has a 0.143 cm 2membrane area and large apical and basolateral access ports for feeding and sampling.The HTS T ranswell-24 culture system is available with a treated polycarbonate membrane in either 0.4 µm or 3.0 µm pore sizes and provides an excellent sub-strate for cell attachment, growth, and differentiation. An open culture reservoir plate is used to reduce liquid handling during cell feeding (medium can be exchanged all at once). Once the cell layers are confluent, the HTS T ranswell-24 insert is transferred to a standard Corning ®24 well microplate for running experiments. Snapwell ™Inserts The Snapwell insert is a modified T ranswell culture insert that contains a 12 mm diameter tissue culture treated polycarbonate or clear polyester mem-brane supported by a detachable ring.These inserts are primarily used for transport and electrophysiological studies.Once cells are grown to confluence, this ring-supported membrane can be placed into either vertical or horizontal diffusion or Using chambers. Chambers are avail-able from Harvard Apparatus: ing Transwell Permeable Supports Helpful Hints 1.Cell morphology and cell densities on permeable supports are influenced by filter pore rger pore sizes may permit some cell types to migrate through the pores on the permeable support.3.Cells grown on permeable supports are often sensitive to initial seeding densi-ty for good cell attachment. On first use, try bracketing a range of seeding densities for optimum growth.4.Cell attachment and spreading may be improved by preincubating permeable supports in medium prior to seeding.5.Cells requiring extracellular matrix coatings on plastic substrates will also require them on permeable supports.6.The T ranswell-Clear insert contains a transparent tissue culture treated polyester membrane that allows easy viewing of cells using phase contrast microscopy.The HTS-96 System is ideal for high throughput transportstudies.HTS Transwell Systems are designed for use withrobotics.Snapwell inserts are designed for use with diffusion or Ussing chambers.7.The T ranswell ®-COL insert contains a PTFE membrane that has been treated with an equimolar mixture of types I and III bovine placental collagens.This results in a biologically stabilized collagen matrix covering the fibrils of the filter membrane. These T ranswell inserts are excellent for the growth of cells requiring a biological coating.General Directions for Use 1.T ranswell inserts are used by first adding medium to the multiple well plate well, then adding the T ranswell insert, and then adding the medium and cells to the inside compartment of the T ranswell insert. Recommended medium volumes are shown in T able 5. 2.An initial equilibrium period may be used to improve cell attachment by adding medium to the multiple well plate well and then to the T ranswell insert. The plate should then be incu-bated for at least one hour or even overnight at the same temperature that will be used to grow the cells. The cells are then added in fresh medium to the T ranswell insert and returned to the incubator.3.The medium level can be checked periodically and fresh medium added as required.4.T ranswell inserts have three openings for standard pipette tips to allow sam-ples to be added or removed from the lower compartment.Add the medium to the culture plate first,then add the medium and cells tothe Transwell insert.The three side wall openings for pipette tip access can be seen in this 24 mm poly-carbonate membrane Transwell insert.The porous bottom of the insert provides independent access to both sides of a cell monolayer giving researchers a versatile tool to study cell transport and other metabolic activities in vitro.5.Cell monolayers may be fixed and stained while in the T ranswell® insert using standard cytological techniques.Avoid using solvents that dissolve poly-styrene or the polycarbonate or poly-ester membrane materials. Processing steps may be carried out by sequential-ly moving the T ranswell insert through a series of multiple well plate wells containing the appropriate reagents.Protocols for fixing and staining T ranswell inserts are available on the Corning Life Sciences web site.6.If it is necessary to remove cells from T ranswell membranes, we recommend rinsing both the T ranswell insert and the plate well. Then the dissociating solution should be added to both the well and the T ranswell insert and incu-bated until the cells begin to come off.A protocol, Trypsinization Procedure for Transwell ®Inserts , for removing cells from T ranswell inserts is available in the technical section of the Corning Life Sciences web site.7. Corning recommends using a Micromatic 8-channel Aspirator (Corning Cat. No. 3389) for removing medium from HTS Transwell-96systems. These aspirators are designed to remove medium and solutions from the upper wells without damaging the sensitive cell monolayers.8.The polycarbonate or polyester mem-brane with the fixed and stained cells attached may be removed from the T ranswell insert by carefully cutting around the membrane edges with a scalpel.9.The collagen-coated PTFE membrane is fragile and requires careful handling during removal. A wetted cellulosic membrane filter should be placed in direct contact with the underside of the T ranswell insert membrane before it is cut out with a scalpel. The wetted,more rigid, cellulosic filter will serve as a support for the collagen-coated membrane.Technical Assistance For additional product or technical infor-mation, please visit /lifesciences or call 1.800.492.1110.Customers outside the United States,please call at 1.978.635.2200.The distance between the tips of the Micromatic ™8-Channel Aspirator (Corning Cat.No.3389) and the membrane layer of the HTS Transwell-96 insert has been optimized to prevent damage to the cell monolayerduring medium removal.Fixed and crystal violet stained CHO-K1 cells on a 3 µm PET membraneOrdering Information Polyester (PET) Membrane Transwell ®-Clear Inserts T ranswell-Clear inserts feature a thin, microscopically transparent polyester membrane that is tissue culture treated for optimal cell attachment and growth. T ranswell-Clear inserts provide excellent cell visibility under phase contrast microscopy and allow assess-ment of cell viability and monolayer formation. T ranswell-Clear inserts are available sterile and preloaded in 6, 12 and 24 multiple well plates. All plates come with lids.Cat. Membrane Growth Surface Membrane Inner Inserts/ No.Diameter* (mm)Area* (cm 2)Pore Size (µm)Packaging Case 3470 6.50.330.412 inserts/24 well plate 243472 6.50.33 3.012 inserts/24 well plate 24346012 1.120.412 inserts/12 well plate 24346212 1.12 3.012 inserts/12 well plate 24345024 4.670.4 6 inserts/6 well plate 24345224 4.67 3.0 6 inserts/6 well plate 24*Values are reported as nominal and may vary due to inherent variability of the manufacturing process. T o insure success, we recommend that researchers validate their methods independent from our reported values.Collagen-Coated Transwell-COL Inserts T ranswell-COL inserts have a transparent, collagen-treated PTFE membrane that promotes cell attachment and spreading and allows cells to be visualized during culture.The T ranswell-COL membrane has an equimolar mixture of types I and III collagen derived from bovine placentas. Unlike traditional coating techniques that result in occluding film layers, Corning’s proprietary coating process results in a biologically stable collagen that covers every fibril of the filter matrix, thereby retaining the porosity of the membrane. T ranswell-COL inserts are sterile and individually blister packed.Appropriate multiple well plates are included in each case. All plates come with lids.Membrane Growth Membrane Multiple Cat. Diameter* Surface Pore Size Inner Well Inserts/ No.(mm)Area* (cm 2)(µm)Packaging Plate Case 3495 6.50.330.4Individually wrapped 2-24 well 243496 6.50.33 3.0Individually wrapped 2-24 well 24349312 1.120.4Individually wrapped 2-12 well 24349412 1.12 3.0Individually wrapped 2-12 well 24349124 4.670.4Individually wrapped 4-6 well 24349224 4.67 3.0Individually wrapped 4-6 well 24*Values are reported as nominal and may vary due to inherent variability of the manufacturing process. T o insure success, we recommend that researchers validate their methods independent from our reported values.24 mm Transwell-Clear InsertPolycarbonate Membrane Transwell ®Inserts These T ranswell inserts feature a thin, translucent polycarbonate membrane available in six pore sizes ranging from 0.1 µm to 12.0 µm. All are treated for optimal cell attachment.They are supplied sterile and come preloaded in multiple well plates or dishes. The polycarbonate membrane is compatible with most organic fixatives and stains. All plates come with lids.Membrane Growth Membrane Cat.Diameter* Surface Pore Size Inner Inserts/ No.(mm)Area* (cm 2)(µm)Packaging Case 3413 6.50.330.412 inserts/24 well plate 483415 6.50.33 3.012 inserts/24 well plate 483421 6.50.33 5.012 inserts/24 well plate 483422 6.50.338.012 inserts/24 well plate 48340112 1.120.412 inserts/12 well plate 48340212 1.12 3.012 inserts/12 well plate 48340312 1.1212.012 inserts/12 well plate 48341224 4.670.4 6 inserts/6 well plate 24341424 4.67 3.0 6 inserts/6 well plate 24342824 4.678.0 6 inserts/6 well plate 24341975440.4 1 insert/100 mm dish 1234207544 3.0 1 insert/100 mm dish 12*Values are reported as nominal and may vary due to inherent variability of the manufacturing process. T o insure success, we recommend that researchers validate their methods independent from our reported values.Snapwell ™Inserts The Snapwell insert is a modified T ranswell culture insert that contains a 12 mm diameter tissue culture treated membrane supported by a detachable ring. Once cells are grown to confluence, this ring-supported membrane can be placed into either ver-tical or horizontal diffusion or Ussing chambers. Chambers are available from Harvard Apparatus: . Snapwell inserts are provided sterile and pre-loaded in 6 well plates. All plates come with lids.Membrane Growth Membrane Cat. Diameter Surface Pore Size Membrane Inner Inserts/No.(µm)*Area* (cm 2)(µm)Material Packaging Case 340712 mm 1.120.4Polycarbonate 6 inserts/6 well plate 24380112 mm 1.120.4Clear Polyester 6 inserts/6 well plate 24380212 mm 1.12 3.0Polycarbonate 6 inserts/6 well plate 24*Values are reported as nominal and may vary due to inherent variability of the manufacturing process. T o insure success, we recommend that researchers validate their methods independent from our reported values.Snapwell Inserts withpolycarbonate (lower) and polyester (upper) membranesHTS Transwell ®-24Systems The HTS T ranswell-24 System has an array of 24 wells with permeable inserts connected by a rigid, robotics-friendly tray that enables all 24 T ranswell supports to be handled as a single unit. The individually packaged product consists of two individually wrapped HTS T ranswell-24 units loaded into open reservoirs and includes two 24 well plates.The bulk packaged products consist of 12 HTS T ranswell-24 units loaded into 24 well plates only. Open reservoirs can be purchased separately.◗Choice of either 0.4 µm polyester membrane or 0.4 µm and 3.0 µm pore polycarbonate membrane ◗Cell growth area is 0.33 cm 2/well ◗Choice of either individual or bulk packaging ◗HTS T ranswell-24 Systems are all tissue culture treated and sterile Cat. Membrane Pore Size No.Description Material (µm)Qty/Cs 3396HTS T ranswell-24 System: insert tray in a PC 0.42reservoir plate with lid, 1/pack; plus a separate 24 well receiver plate with lid, 1/pack 3379HTS T ranswell-24 System: insert tray in a reservoir PET 0.42plate with lid, 1/pack; plus a separate 24 well receiver plate with lid, 1/pack 3397HTS T ranswell-24 System, Bulk Packed: PC 0.412insert trays in 24 well plates with lids, 12/pack 3378HTS T ranswell-24 System, Bulk Packed: insert trays PET 0.412in 24 well plates with lids, 12/pack 3398HTS T ranswell-24 System: insert tray in a PC 3.02reservoir plate with lid, 1/pack; plus a separate 24 well receiver plate with lid, 1/pack 3399HTS T ranswell-24 System, Bulk Packed: PC 3.012insert trays in 24 well plates with lids, 12/pack 3395HTS T ranswell-24 Reservoir (Feeder) Plate and lid, NA NA 48not treated, 12/packHTS Tranwell-24 System showing both the culture reservoir and the 24 well microplate.HTS Transwell ®-96 SystemsThe HTS T ranswell-96 System has an array of 96 wells with permeable inserts connected by a rigid, robotics-friendly tray that enables all 96 inserts to be handled as a single unit.Each HTS T ranswell-96 System includes 1 integral tray containing 96 individual inserts in a reservoir plate (no wells) with a removable media stabilizer for cell growth steps,plus a 96 well receiver plate for growth or assay steps and 2 lids.◗HTS T ranswell-96 insert membranes are all tissue culture treated and sterile ◗Choice of either a 0.4 µm polyester membrane or 0.4 µm and 3.0 µm pore poly-carbonate membranes◗Cell growth area is 0.143 cm 2/well which is 20 to 50% greater than competitive devices ◗Large apical and basolateral access ports for easier filling and sampling ◗Removable media stabilizer reduces media spills during handling◗Automation optimized design with multichannel feeder ports, improved gripping surface and optional bar coding◗Corning offers the Micromatic ™8-Channel Aspirator (Corning Cat. No. 3389) to help safely vacuum aspirate medium or buffers from the apical portion of the HTS T ranswell-96 inserts.Cat. Membrane Pore Size No.Description Material (µm)Qty/Cs3380HTS T ranswell-96 System: insert tray in aPET 1.01reservoir plate with lid 1/pack; plus a separately packed 96 well receiver plate with lid, 1/pack3392HTS T ranswell-96 System, Bulk Packed: insert trays PET 1.05in a reservoir plates with lids, 5/pack; plus separately packed 96 well receiver plates with lids, 5/pack3381HTS T ranswell-96 System: insert tray in a reservoir PC 0.41plate with lid, 1/pack; plus a separately packed 96 well receiver plate with lid, 1/pack3391HTS T ranswell-96 System, Bulk Packed: insert trays PC 0.45in reservoir plates with lids, 5/pack; plus separately packed 96 well receiver plates with lids, 5/pack 3382HTS T ranswell-96 Receiver Plate with lid, NA NA 10not treated, 10/pack3383HTS T ranswell-96 Reservoir (Feeder) Plate with NANA10removable media stabilizer and lid, not treated, 10/pack3389Micromatic 8-Channel Aspirator for NA NA 1HTS T ranswell-96 Systems, AutoclavableHTS Tranwell-96 Systemshowing the culture reservoir with removable media stabilizer (top),the 96 well insert tray (middle) and the 96 well receiver plate(bottom).Using the Micromatic8-Channel Aspirator (Corning Cat.No.3389)reduces cell death ordetachment caused by too rapid removal of solutions or media during vacuum aspiration.6,12,and 24 Well Cell Culture PlatesThese multiple well plates are treated for optimal cell attachment, sterilized by gamma radiation and are certified nonpyrogenic. All plates have a uniform footprint and a raised bead to aid in stacking. Alphanumeric labels provide individual well identification. The 6.5, 12, and 24.5 mm T ranswell inserts are designed to automatically center themselves when placed into the appropriate culture plate.Cat. Number Well GrowthNo.of Wells Diameter* (mm)Surface Area* (cm 2)Qty/PkQty/Cs3506634.89.551003516634.89.515035121222.1 3.8510035131222.1 3.815035272415.6 1.9510035132415.6 1.9150*Values are reported as nominal and may vary due to inherent variability of the manufacturing process. T o insure success, we recommend that researchers validate their methods independent from our reported values.Corning and Transwell are registered trademarks of Corning Incorporated,Corning,NY.Discovering Beyond Imagination,Flame of Discovery design,and Snapwell are trademarks of Corning Incorporated,Corning,NY.Micromatic is a registered trademark of Popper and Sons,New Hyde Park NY.Corning Incorporated,One Riverfront Plaza,Corning,NY 14831-0001© 2005 C o r n i n g I n c o r p o r a t e d P r i n t e d i n U S A 5/05 P O D C L S -C C -007W R E V 2Corning Incorporated Life Sciences45 Nagog Park Acton,MA 01720t 800.492.1110t 978.635.2200f /lifesciencesWorldwide Support OfficesA S I A Australiat 61 2-9416-0492f 61 2-9416-0493Chinat 86 21-3222-4666f 86 21-6288-1575Hong Kong t 852-2807-2723f 852-2807-2152Indiat 91 11 341 3440f 91 11 341 1520Japant 81 (0) 3-3586 1996/1997f 81 (0) 3-3586 1291/1292Koreat 82 2-796-9500f 82 2-796-9300Singapore t 65 6733-6511f 65 6861-2913Taiwant 886 2-2716-0338f 886 2-2716-0339E U R O P E Francet 0800 916 882f 0800 918 636Germanyt 0800 101 1153f 0800 101 2427The Netherlands & All OtherEuropean Countries t 31 (0) 20 659 60 51f 31 (0) 20 659 76 73United Kingdom t 0800 376 8660f 0800 279 1117L AT I N A M E R I C A Brasilt (55-11) 3089-7419f (55-11) 3167-0700Mexicot (52-81) 8158-8400f (52-81) 8313-8589For additional product or technical information, please visit /lifesciences or call 800.492.1110. Customers outside the United States, please call +1.978.635.2200or contact your local Corning sales office listed below.Corning offers a variety of multiple well plate designs and sizes.。

P0033 细胞膜蛋白与细胞浆蛋白提取试剂盒

细胞膜蛋白与细胞浆蛋白抽提试剂盒产品简介：碧云天的细胞膜蛋白与细胞浆蛋白抽提试剂盒(Membrane and Cytosol Protein Extraction Kit)提供了一种比较简单、方便地从培养细胞或组织中抽提细胞膜蛋白和细胞浆蛋白的方法。

抽提的膜蛋白不仅包括质膜上的膜蛋白，也包括线粒体膜、内质网膜和高尔基体膜等上的膜蛋白。

本试剂盒通过匀浆适度破碎细胞，经低速离心去除细胞核和少数未破碎的细胞产生的沉淀，随后取上清高速离心获得细胞膜沉淀和含有细胞浆蛋白的上清，然后通过优化的膜蛋白抽提试剂从沉淀中抽提获取膜蛋白。

约90分钟即可完成培养细胞或组织的细胞膜蛋白与细胞浆蛋白的分离和抽提。

抽提得到的蛋白可以用于SDS-PAGE，Western、酶活性测定等后续实验。

膜蛋白抽提试剂中含有蛋白酶抑制剂、磷酸酯酶抑制剂和EDTA等，后续不适合用于蛋白酶、磷酸酯酶等受这些抑制剂影响的酶的活性测定，但抽提获得的膜蛋白或细胞浆蛋白适合用于检测蛋白的磷酸化水平。

本试剂盒按照本说明书的操作步骤可以抽提100个细胞或组织样品。

保存条件：-20℃保存，一年有效。

注意事项：需自备PMSF。

PMSF一定要在抽提试剂加入到样品中前2-3分钟内加入，以免PMSF在水溶液中很快失效。

PMSF(ST506)可以向碧云天订购。

使用本试剂盒抽提到的细胞膜蛋白与细胞浆蛋白均可直接用碧云天生产的BCA法蛋白浓度测定试剂盒(P0009/P0010/P0010S/P0011/P0012/P0012S)测定蛋白浓度。

抽提获得的细胞膜蛋白不适合用Bradford法测定蛋白浓度。

为了您的安全和健康，请穿实验服并戴一次性手套操作。

使用说明：1.准备试剂：室温融解并混匀膜蛋白抽提试剂A和B，融解后立即置于冰浴上。

取适量的膜蛋白抽提试剂A和B备用，在使用前数分钟内加入PMSF，使PMSF的最终浓度为1mM。

2.准备细胞或组织样品：a. 对于细胞(1) 收集细胞对于贴壁细胞：培养约2000-5000万细胞，用PBS洗一遍，用细胞刮子刮下细胞或用含有EDTA但不含胰酶的细胞消化液处理细胞使细胞不再贴壁很紧，并用移液器吹打下细胞。

上颌窦提升术骨替代材料的研究进展

上颌窦提升术骨替代材料的研究进展高萍;王旭霞【摘要】上颌后牙区解剖结构的特殊性导致了上颌后牙缺失后垂直骨量丧失严重,限制了上颌后牙区口腔种植的适应证,增加了种植手术的风险,成为临床上种植义齿修复的主要障碍.上颌窦提升术可以改善萎缩性上颌骨的牙槽嵴高度,而植骨材料的应用可充分解决上颌后部种植骨量不足的问题.本文介绍了上颌窦提升术中,骨替代材料的研究应用情况.%Ridge resorption is the consequence of tooth loss. When the residual bone volume is too diminished and direct replacement of the missing tooth with a dental implant is unsuitable, it may be necessary to augment the deficient ridge prior to dental implant placement. Various reconstructive materials to augment the alveolar bone ridge for osteointegrated dental implants are widely used. Non-vascularized bone, allogenic bone, and artificial bone substitutes are used in sinus lift to segment the sinus floor. This paper describes the recent advances of bone substitute materials in maxillary sinus lift surgery.【期刊名称】《口腔颌面外科杂志》【年(卷),期】2017(027)004【总页数】4页(P295-298)【关键词】上颌窦提升;骨替代材料;种植【作者】高萍;王旭霞【作者单位】山东大学口腔医学院口腔颌面外科,山东济南 250012;山东大学口腔医学院口腔颌面外科,山东济南 250012【正文语种】中文【中图分类】R782.1上颌后牙的缺失，牙槽骨不断的丧失以及上颌窦腔的气化，导致上颌窦不断扩大及上颌后牙牙槽骨宽度和高度明显下降，从而增加了种植手术的风险，限制了上颌后牙区口腔种植的适应证[1]，而上颌窦底提升术（maxillary sinus augmentation，MSA）是上颌磨牙区骨量不足的常规治疗方法。

Elabscience

（本试剂盒仅供体外研究使用，不用于临床诊断！）Elabscience®还原型谷胱甘肽(GSH)比色法测试盒Reduced Glutathione (GSH) Colorimetric Assay Kit产品货号：E-BC-K030-S产品规格：50 assays(48 samples)/100 assays(96 samples)检测仪器：紫外-可见光分光光度计（420 nm）使用前请仔细阅读说明书。

如果有任何问题，请通过以下方式联系我们：销售部电话************，************技术部电话131****6790具体保质期请见试剂盒外包装标签。

请在保质期内使用试剂盒。

联系时请提供产品批号(见试剂盒标签)，以便我们更高效地为您服务。

用途本试剂盒适用于检测血清、血浆、动植物组织样本及培养细胞中GSH的含量。

检测原理还原型谷胱甘肽（GSH）可与二硫代二硝基苯甲酸（DTNB）反应产生硫代硝基苯甲酸和谷胱甘肽二硫化物（反应式见下图），硝基巯基苯甲酸是一种黄色化合物，在420 nm处，可进行比色定量测定还原型谷胱甘肽（GSH）的含量。

本试剂盒检测组织和细胞样本时，需测定总蛋白浓度，推荐使用考马斯亮蓝法（货号：E-BC-K168-S）提供试剂和物品说明：试剂严格按上表中的保存条件保存，不同测试盒中的试剂不能混用。

对于体积较少的试剂，使用前请先离心，以免量取不到足够量的试剂。

所需自备物品仪器：紫外-可见光分光光度计（420 nm）、涡旋混匀仪、磁力搅拌器、微量移液器（1000 μL，200 μL，100 μL，10 μL）、烧杯（250 mL）耗材：枪头（1000 μL，200 μL，10 μL ）、EP管（5 mL、2mL）、吸水纸、擦镜纸、磁力搅拌子试剂：双蒸水或去离子水、生理盐水（0.9% NaCl）或PBS（0.01 M，pH 7.4）。

试剂准备①检测前，试剂盒中的试剂平衡至室温。

易静-cellmembrane

异常PrP*有传染性，使其它正常PrP 变成异常PrP*
正常PrP蛋白
引起疯牛病的异常PrP*蛋白
The Nobel Prize in Physiology or Medicine 1997
"for his discovery of Prions - a new biological principle of infection"
1. helix
Figure 11-28 Essential Cell Biology (© Garland Science 2010)
2. barrel
Figure 11-25 Essential Cell Biology (© Garland Science 2010)
3. The structures versus the functions
PhospholipidsБайду номын сангаасwith a amino acid head
Membrane proteins
1. The ways membrane proteins associate with the lipid bilayer
2. The structures of membrane proteins 3. The structures versus the functions
Myelin of the sciatic nerve
Inner membrane of the mitochondrion
History of discovery
1890’ ，Overton：root absorption， Found: the more fat-soluble, the easier to be absorbed. －The membrane is composed of lipids ？

单羧酸转运蛋白1促进胰腺导管癌细胞浸润转移的机制研究

《中国癌症杂志》2019年第29卷第3期 CHINA ONCOLOGY 2019 Vol.29 No.3183欢迎关注本刊公众号·论　著·基金项目：山东省医药卫生科技发展计划项目(2015WS0211)；山东省自然科学基金项目(ZR2014HM051)。

通信作者：孙　超 E-mail: 347754658@单羧酸转运蛋白1促进胰腺导管癌细胞浸润转移的机制研究张　敏1，王琳娜2，孙　超31.济南市人民医院检验科，山东济南 271100；2.山东省青岛疗养院检验科，山东青岛 266071；3.青岛市市立医院高压氧科，山东青岛 266071［摘要］背景与目的：单羧酸转运蛋白1（monocarboxylate transporter 1，MCT1）是细胞转运乳酸、丙酮酸等代谢产物及能量物质的一种重要蛋白质，其在胰腺导管癌中的作用及机制鲜有研究报道。

该研究旨在探讨MCT1在胰腺导管癌中的表达及临床病理学意义。

方法：纳入78例胰腺导管癌患者的癌组织及癌旁正常组织，运用免疫组织化学技术检测MCT1在癌组织和癌旁正常组织中的表达水平并分析其临床病理学意义。

在体外细胞系水平上，我们运用胰腺癌细胞系PANC-1和Capan-1，运用细胞克隆形成实验、细胞划痕和Transwell 实验分析沉默MCT1后胰腺癌细胞增殖、迁移和浸润的改变。

为明确MCT1的相关作用机制，我们通过生物信息学分析，预测miR-124-3p 是MCT1的潜在调控微小RNA ；为了进一步验证，我们运用双荧光素酶报告实验分析miR-124-3p 对MCT1的调控效果；运用实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction ，RTFQ-PCR ）分别检测51对新鲜胰腺癌组织中MCT1和miR-124-3p 的基因表达并分析两者的相关性。

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Published Ahead of Print 30 March 2009. 10.1128/AAC.01682-08.2009, 53(6):2312. DOI:Antimicrob. Agents Chemother. BayerRubio, Cynthia C. Nast, Michael R. Yeaman and Arnold S. Nagendra N. Mishra, Soo-Jin Yang, Ayumi Sawa, AileenStaphylococcus aureus Strains of Methicillin-Resistant of In Vitro-Selected Daptomycin-Resistant Analysis of Cell Membrane Characteristics /content/53/6/2312Updated information and services can be found at: These include:REFERENCES/content/53/6/2312#ref-list-1at: This article cites 46 articles, 33 of which can be accessed free CONTENT ALERTSmore»articles cite this article), Receive: RSS Feeds, eTOCs, free email alerts (when new /site/misc/reprints.xhtml Information about commercial reprint orders: /site/subscriptions/To subscribe to to another ASM Journal go to: on June 26, 2014 by guest/Downloaded fromA NTIMICROBIAL A GENTS AND C HEMOTHERAPY,June2009,p.2312–2318Vol.53,No.6 0066-4804/09/$08.00ϩ0doi:10.1128/AAC.01682-08Copyright©2009,American Society for Microbiology.All Rights Reserved.Analysis of Cell Membrane Characteristics of In Vitro-Selected Daptomycin-Resistant Strains of Methicillin-ResistantStaphylococcus aureusᰔNagendra N.Mishra,1Soo-Jin Yang,1Ayumi Sawa,1Aileen Rubio,2Cynthia C.Nast,3,4Michael R.Yeaman,1,4and Arnold S.Bayer1,4*Division of Infectious Diseases,Los Angeles Biomedical Research Institute at Harbor—University of California at Los Angeles(UCLA)Medical Center,1124West Carson St.,Torrance,California905021;Cubist Pharmaceuticals, Lexington,Massachussetts2;Cedars-Sinai Medical Center,Los Angeles,California3;andDavid Geffen School of Medicine at UCLA,Los Angeles,California4Received19December2008/Returned for modiﬁcation15February2009/Accepted21March2009Our previous studies of clinical daptomycin-resistant(Dap r)Staphylococcus aureus strains suggested thatresistance is linked to the perturbations of several key cell membrane(CM)characteristics,including the CMorder(ﬂuidity),phospholipid content and asymmetry,and relative surface charge.In the present study,weexamined the CM proﬁles of a well-known methicillin-resistant Staphylococcus aureus(MRSA)strain(MW2)after in vitro selection for DAP resistance by a20-day serial passage in sublethal concentrations of DAP.Compared to levels for the parental strain,Dap r strains exhibited(i)decreased CMﬂuidity,(ii)the increasedsynthesis of total lysyl-phosphatidylglycerol(LPG),(iii)the increasedﬂipping of LPG to the CM outer bilayer,and(iv)the increased expression of mprF,the gene responsible for the latter two phenotypes.In addition,wefound that the expression of the dlt operon,which also increases positive surface charge,was enhanced in theDap r mutants.These phenotypic and genotypic changes correlated with reduced DAP surface binding,mir-roring observations made in clinical Dap r isolates.In this strain,serial exposure to DAP induced an increasein vancomycin MICs into the vancomycin-intermediate S.aureus(VISA)range(4␮g/ml)in parallel withincreasing DAP MICs.Also,this Dap r strain exhibited signiﬁcantly thicker cell walls than the parental strain,potentially correlating with the coevolution of the VISA phenotype and implicating cell wall structure and/orfunction in the Dap r phenotype.Importantly,despite the overexpression of mprF and dlt,the relative netpositive surface charge was decreased in the Dap r mutants,suggesting that other factors contribute to thesurface charge alterations and that a simple charge repulsion mechanism could not entirely explain the Dap rphenotype in these strains.Staphylococcus aureus can cause a variety of localized and more invasive infections,especially when the skin or a mucosal barrier is breached(26).Such infections can be particularly problematic when the organism is multiply drug resistant,as in the case of methicillin-resistant S.aureus(MRSA).The poor treatment outcomes often associated with serious MRSA in-fections demonstrates the need for alternative effective treat-ment options.Daptomycin(DAP)is a lipopeptide antibiotic that was approved for the treatment of gram-positive bacterial infections in2003(12,14,17).The agent has received much interest because of its activity against multidrug-resistant, gram-positive bacteria such as MRSA,vancomycin-resistant enterococci,and glycopeptide-intermediate and-resistant S. aureus(14,43).While the mode of action of DAP is not entirely clear(30),it is known that calcium-dependent binding to the bacterial CM and the disruption of CM function(39)are critically involved.Although the native DAP molecule is an-ionic,its active,calcium-decorated form renders it a de facto cationic antimicrobial peptide(CAP)(14,43).Despite the general acceptance of the role of CM targeting in the lethal mechanisms of DAP,the relative contributions of the CM,the cell wall(CW),and other targets beyond these sites(e.g., intracellular targets)have not been clearly established(44). The objective of our study was to further elucidate changes in cell surface phenotypes relevant to Dap r,including CMﬂuid-ity,fatty acid content,phospholipid(PL)composition and asymmetry,and surface charge,in a well-known MRSA strain (MW2)after the in vitro selection of DAP resistance by serial passage in sublethal DAP concentrations.(This work was presented in part at the48th Annual Inter-science Conference on Antimicrobial Agents and Chemother-apy/Infectious Diseases Society of America46th Annual Meet-ing,Washington,DC,25to28October2008[abstr.C1-177],a joint meeting of the American Society for Microbiology and the Infectious Diseases Society of America.)MATERIALS AND METHODSBacterial strains.All strains used in this study are listed in Table1.The parental strain,MW2,is of the USA400pulsotype.The details of the20-day serial passage selection process have been previously reported(9).The selective pressure of growth in sublethal concentrations of DAP resulted in the progres-sive accumulation of single-nucleotide polymorphisms(SNPs)over time that correlated with incremental decreases in DAP susceptibility.The majority of the SNPs resulted in amino acid substitutions(9).The loci affected include mprF, encoding lysyl-phosphatidylglycerol(LPG)synthase and translocase(9,33);*Corresponding author.Mailing address:LA Biomedical Research Institute at Harbor-UCLA,1124West Carson Street,Bldg.RB2, Room225,Torrance,CA90502.Phone:(310)222-6422.Fax:(310) 782-2016.E-mail:abayer@.ᰔPublished ahead of print on30March2009.2312 on June 26, 2014 by guest / Downloaded fromyycG ,encoding a histidine kinase of an operon involved in both CW and CM metabolism (9,27);and rpoB ,the ␤subunit of RNA polymerase (9).The MICs of DAP,oxacillin,and vancomycin were determined by using standard proce-dures for the Etest (AB Biodisc,Dalvagen,Sweden)on Mueller-Hinton agar (MHA)plates (Difco Laboratories,Detroit,MI).For the DAP Etest,plates were supplemented with calcium (50␮g/ml CaCl 2).Growth curves (24h)of the strains in tryptic soy broth (TSB)revealed that the more Dap r passage strains (CB2203and CB2205)grew modestly slower than the parental strain (data not shown).However,all six strains reached equivalent counts at 24h (ϳ5ϫ109CFU/ml).Population analysis.The population analysis of a selected strain pair (the parent strain CB1118and the 20-day Dap r passage mutant CB2205)was carried out with DAP,oxacillin,and vancomycin by following a standard agar dilution protocol as previously described (28).DAP population analyses were performed on calcium-supplemented (50␮g/ml)MHA plates.CAP susceptibilities.Our prior studies showed that Dap r clinical strains ex-hibited cross-reductions in susceptibility to prototypical mammalian host defense CAPs of white blood cell (hNP-1)or platelet (tPMP-1)origin (12).To examine this phenomenon in the current laboratory-derived Dap r strain set,we carried out in vitro susceptibility assays with the same two CAPs.Standard MICs for such CAPs are not routinely performed,since conventional nutrient medium tends to mitigate the activity of these peptides;thus,bactericidal assays were carried out (47).For tPMP-1,a range of 0.5to 2␮g/ml was used.For hNP-1,a concentration of 20␮g/ml was used.tPMP-1was prepared as previously described (46);hNP-1was purchased from Peptide International (Louisville,KY).Brieﬂy,log-phase S.aureus cells were diluted into the test CAP solutions to achieve the desired ﬁnal inoculum (103and 105CFU/ml for tPMP-1and hNP-1,respectively)(44,45)and then incubated at 37°C.At 120min of exposure,samples were removed and processed for quantitative culture to assess the extent of killing by each CAP.Final data were expressed as the means Ϯstandard deviations (SD)of surviving log 10CFU/milliliter.CM ﬂuidity.Fluidity measurements were carried out using the ﬂuorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH).The protocol for DPH incorpora-tion into target CMs,the measurement of ﬂuorescence polarization,and the calculation of the degree of ﬂuorescence polarization (polarization index [PI])are described in detail elsewhere (a Biotek Model SFM 25spectroﬂuorimeter was used;excitation and emission wavelengths were 360and 426nm,respec-tively,for DPH)(2).An inverse relationship exists between PI values and CM ﬂuidity (i.e.,a lower PI equates to a greater extent of CM ﬂuidity)(2).These assays were performed a minimum of 10times for each strain on separate days.CM fatty acid composition.Fatty acids of total lipids extracted from S.aureus CMs were analyzed using gas-liquid chromatography after conversion to their methyl ester form as previously described in detail (courtesy of Microbial ID Inc.,Newark,DE)(2).Cell surface charge.Poly-L -lysine (PLL)is a polycationic molecule that is used to study the interactions of CAPs with charged CM bilayers (10,16).A ﬂuores-cein isothiocyanate-labeled PLL binding assay was performed using ﬂow cyto-metric methods as previously described (using a FACSCalibur;Beckman Instru-ments,Alameda,CA)(29).Data were expressed as mean ﬂuorescent units (ϮSD).The lower the amount of residual cell-associated label,the greater thePLL repulsion and the more positively charged the S.aureus cell envelope (29).At least three independent runs were performed on separate days.CW thickness.CW thickness was determined by transmission electron micros-copy (EM).Cells were prepared for EM by previously published techniques (21).One hundred CW thickness measurements were taken from a minimum of 50bacterial cells for each strain at a magniﬁcation of 190,000ϫ(model 100CX;JEOL,Tokyo,Japan)using digital image capture and morphometric measure-ment (v54;Advanced Microscopy Techniques,Danvers,MA).The bacterial cells examined each were in a different ﬁeld,were intact,were in full cross-section without a sectioning artifact,and were nondividing.CWs were measured from the outer aspect of the CM to the outer aspect of the CW.Mean (ϮSD)CW thickness then was determined for each strain.Isolation of RNA.For RNA isolation,fresh overnight cultures of S.aureus strains were used to inoculate TSB to an optical density at 600nm of 0.1.Cells were harvested during exponential growth (2h),early stationary phase (6h),and late stationary phase (12h).Total RNA was isolated from the cell pellets by using the RNeasy kit (Qiagen,Valencia,CA)and the FASTPREP FP120in-strument (BIO 101,Vista,CA)according to the manufacturers’recommended protocols.Northern blot analysis.Northern blot analyses were performed as described previously,with minor modiﬁcations (35).Digoxigenin-labeled DNA probes were synthesized using a PCR-based digoxigenin probe synthesis kit (Roche)using primers mprF-F (5Ј-GTAGTAATCACATTGTATCGGGAGT-3Ј),mprF-R (5Ј-GATGCATCGAAAACATGGAATAC-3Ј),dlt-F (5Ј-ATATGATT GTTGGGATGATTGGTGCCA-3Ј),and dlt-R (5Ј-ACATATGGTCCAACTG AAGCTACG-3Ј)to synthesize mprF-and dlt-speciﬁc probes,respectively.CM PL content and PL asymmetry measurements.PLs were extracted from S.aureus by standard methods (7,29).The three major S.aureus PLs,phosphati-dylglycerol (PG;negatively charged),cardiolipin (CL;negatively charged),and LPG (positively charged),were separated by two-dimensional thin-layer chro-matography using Silica 60F254HPTLC plates (Merck).First-dimension chlo-roform-methanol–25%ammonium hydroxide (65:25:6,by volume)in the vertical orientation and second-dimension chloroform:water:methanol:glacial acetic acid:acetone (45:4:8:9:16,by volume)in the horizontal orientation were used for the separation of the PLs for further quantitation by phosphate estimation (29).For quantitative analysis,isolated PLs were digested at 180°C for 3h with 0.3ml 70%perchloric acid and quantiﬁed spectrophotometrically at an optical density at 600nm as previously described (29).At least three independent runs were performed on separate days.Fluorescamine,which speciﬁcally labels only surface-exposed amino-PLs (i.e.,outer CM leaﬂet),was used to assess the CM LPG distribution in the outer and inner CM bilayers.The protocols of labeling and the quantitative estimation of PL asymmetry by UV detection,ninhydrin spraying,and chemical analysis were strictly followed as previously described (12,29).Statistical analysis.Data were analyzed by the two-tailed student’s t test,with P Ͻ0.05representing a signiﬁcant difference.RESULTSMIC.The Etest MICs are listed in Table 1.As previously described (9),the DAP MICs progressively increased during the 20-day serial exposure period,from a MIC of 1to 32.The vancomycin MICs increased by one doubling dilution with sublethal DAP passage from a MIC of 2to 4␮g/ml during this same time period.The oxacillin MIC for the parental strain was 24␮g/ml but was 1.5␮g/ml for the 20-day passage isolate,exhibiting the see-saw reciprocal phenomenon previously de-scribed for vancomycin-intermediate S.aureus (VISA)strains for vancomycin and oxacillin (37,38).Population analysis.As expected,the DAP population curve was shifted to the right in the 20-day mutant (CB2205)compared to that of the parental strain (CB1118)(data not shown).This shift corresponds to an increase in the DAP MIC and reduced oxacillin susceptibility in this population of organ-isms.In contrast,the oxacillin population curve was shifted to the left for the mutant (i.e.,more oxacillin susceptible),paral-leling the MIC data described above and conﬁrming a modest reciprocal effect on oxacillin susceptibility (data not shown).TABLE 1.List of strains and their antimicrobial susceptibilityproﬁles to DAP and vancomycinStrainLength of serial passage (days)Mutation(s)MIC (␮g/ml)of:DAPVAN aParental CB1118(MW2)12Passage set CB22011Acetate CoA ligase b1.53CB22025Acetate CoA ligase MprF T345I 33CB220314Acetate CoA ligase MprF T345IRpoB I953S64CB220520Acetate CoA ligase MprF T345IRpoB I953S YycG S221P124a VAN,vancomycin.bNoncoding region.CoA,coenzyme A.V OL .53,2009IN VITRO-SELECTED Dap r STRAINS OF S.AUREUS 2313on June 26, 2014 by guest/Downloaded fromMoreover,the vancomycin population curve of the baseline organism already had a subpopulation of VISA isolates,and these also shifted to the right for the Dap r mutant,which is compatible with its increase in vancomycin MICs into the het-ero-VISA range (Fig.1).CAP susceptibilities.The Dap r mutant (CB2205)showed cross-reductions in susceptibilities to the two prototypical mammalian CAPs.For tPMP-1,the parental strain viability was reduced to below the level of detection during the 2-h exposure to concentrations of 0.5to 2␮g/ml;in contrast,the Dap r mutant exhibited the survival proﬁle (mean percent sur-vival ϮSD)at 0.5,1,and 2␮g/ml tPMP-1,and similarly for hNP-1,the mean percent survival (ϮSD)after 2h of exposure to 20␮g/ml peptide of the parental and Dap r strain 2205is shown in Table 2.CM ﬂuidity.There was a modest,although detectable,de-crease of relative CM ﬂuidity (an increasing PI means increas-ing membrane rigidity)as passage strains (CB2201,CB2202,CB2203,and CB2205)became more Dap r than the parental strain,CB1118.The PIs from 10independent measurements were 0.3219Ϯ0.0339,0.3281Ϯ0.0421,0.3267Ϯ0.0374,0.3288Ϯ0.0272,and 0.3302Ϯ0.0401for the parental strain and four progressively more Dap r strains,respectively.As con-trols for these studies,we included an isogenic strain set,ISP479C (0.3312Ϯ0.0122)and ISP479R (0.3119Ϯ0.0194),which are known to be less and more ﬂuid,respectively (2).Cell surface charge.The relative surface charge values of all strains are represented in Fig.2.As controls for these studies,we used an SA113strain that is known to exhibit a relatively high surface positive charge compared to that of its isogenic ⌬dlt knockout variant (32).Unexpectedly,the PLL binding was observed to be higher in the Dap r strains than in the parental strain.Thus,there was an inverse relationship between DAP resistance and surface charge.CM fatty acid composition.Fatty acid data for the parental strain and the most Dap r strain (i.e.,CB2205)showed a similar pattern in terms of iso and ante-iso (branched chain)fatty acids,unsaturation indices,and acyl chain length proﬁles (data not shown).CW thickness.As shown in Fig.3,the Dap r strain has sig-niﬁcantly thicker CWs (31.3Ϯ2.7nm)than the parental strain (20.4Ϯ2.7nm;P Ͻ0.05).Expression of genes that affect surface charge (mprF and dlt operon).It has been shown that the S.aureus mprF and dlt operon encode proteins that enhance the net positive charge of the cell surface envelope by lysinylating CM PG and alanylat-ing CW teichoic acids,respectively (31,32,33,41).It also has been hypothesized that MprF is responsible for translocating LPG to the outer CM bilayer (23).To determine the transcrip-tional proﬁles of these two genes,Northern blot analyses were performed on RNA samples isolated from cultures of CB1118,CB2201,CB2202,CB2203,and CB2205grown in TSB.As shown in Fig.4A,mprF expression was detected in all ﬁve strains during early exponential and stationary growth phases (2and 12h).Importantly,the increased levels of mprF tran-scripts were detected in Dap r strains (CB2202,CB2203,and CB2205)during early exponential growth (2h).Additionally,mprF expression was increased in all four passage strains com-pared to levels for the parental strain in stationary phase.Similarly to mprF expression,the transcription of the dlt operon was increased in the three Dap r strains during early exponential growth (Fig.4B).CM PL content and asymmetry measurements.Two-dimen-sional thin-layer chromatography and LPG-ﬂuorescamine labeling revealed that the total amounts of the positively charged LPG (outer and inner layers)in the CM were higher in the Dap r strains than in the parental strain.More-over,there was a progressive upward trend in the proportion of total and outer CM leaﬂet LPG in the strain set during the in vitro selection of Dap r strains.The proportions of the other PLs (i.e.,PG and CL)were similar among the strains,although as the proportion of LPG increased in the more Dap r strains,the proportion of PG declined concordantly.Small amounts of CL were detected in these experiments.These data are shown in Table3.FIG.1.Population analyses of study strains upon exposure to a range of vancomycin concentrations (conc.).These data represent the means (ϮSD)for three separate assays.TABLE 2.In vitro susceptibilities of study strains to CAPsStrain%Survival (means ϮSD)after 2-h exposure to:0.5␮g/ml tPMP-11.0␮g/ml tPMP-12.0␮g/ml tPMP-120␮g/ml hNP-1CB1118ND a ND ND 68Ϯ15CB2205105Ϯ2990Ϯ2699Ϯ37105Ϯ26aND,not detected (below the detectionlevel). surface charge for the strain set based on ﬂuorescein-isothiocyanate labeled PLL (PLL*)binding.The higher the intensity in ﬂuorescent units (FUs),the more negative the relative surface charge.2314MISHRA ET AL.A NTIMICROB .A GENTS C HEMOTHER .on June 26, 2014 by guest/Downloaded fromDISCUSSIONIn the context of the rising problems of multidrug resistance among gram-positive pathogens,especially to glycopeptide agents,DAP (lipopeptide)offers an important addition to the antibacterial armamentarium (15).However,examples of the isolation of Dap r S.aureus strains in association with clinical treatment failure (9,12,15)suggest the need for the detailed analysis of the mechanism(s)of resistance of such organisms (12).In the current study,we utilized a well-characterized isogenic MRSA strain set selected for DAP resistance by serial in vitro passage (9)to compare their phenotypic proﬁles to data we recently published related to a clinically derived S.aureus isogenic strain set (12).Since the putative mechanism of action of DAP has focused on CM targeting (12),we charac-terized a number of key CM properties in this study.As de-tailed below,the cadre of cell surface and CM perturbations found in the present laboratory-derived strain set were strik-ingly disparate from those previously documented for Dap r strains isolated from recalcitrant clinical infections (12).Several reports have demonstrated a correlation between decreased oxacillin resistance and a concomitant increase in resistance to vancomycin into the VISA range (the so-called see-saw effect)(37,38).The mechanism(s)involved in this phenomenon appear to be multifactorial.For example,some studies have implicated the deletion of mecA or mecA muta-tions in the presence of vancomycin exposures in the see-saw effect (1,34,38).These investigations have suggested an in-compatibility between the simultaneous expression of high-level vancomycin resistance and high-level methicillin resis-tance in MRSA.PBP2a,a mecA -encoded protein,is not capable of cross-linking peptidoglycan-containing stem peptide modiﬁed by van resistance genes (8,36).Interestingly,we found a similar reciprocal effect,involving increasing DAP resistance and the evolution of the VISA phenotype on the one hand and decreasing oxacillin resistance on the other hand.In contrast,Camargo et al.(4)recently observed that laboratory-derived Dap r strains had increased resistance to vancomycin and increased resistance to oxacillin,suggesting strain-speciﬁc and/or independent mechanisms of resistance to these agents as related to the induction of the reciprocal phenomenon.For example,relevantly to our current ﬁndings,Komatsuzawa et al.(19)described an association between mutations in the mprF (fmtC )gene and oxacillin resistance in vitro.Thus,it seems reasonable to speculate that the gain-in-function mprF SNP as we demonstrated secondarily inﬂuences the full expression of the mecA -mediated oxacillin resistance phenotype.Taking these results together,the relationship of increasing DAP and vancomycin resistance coincident with decreasing oxacillin re-sistance in vitro has the following features:(i)relative strain speciﬁcity,(ii)a nonrequirement for the presence of DAP (in contrast to vancomycin-oxacillin interactions)(1),and (iii)a mutation in mprF as seen in our Dap r strains could well inﬂu-ence the expression of oxacillin resistance.One prominent correlate of resistance to theCM-targetingFIG.3.Transmission EM of the parental strain (CB1118)and Dap r mutant (CB2205).The thickness of the CB1118(20.4Ϯ2.7nm)and CB2205(31.3Ϯ2.7nm)CWs isindicated.FIG.4.(A and B)Northern blot analyses for mprF and dlt operon expression.Total cellular RNA samples were isolated at 2,6,and 12h postinoculation from S.aureus cells cultured in TSB.V OL .53,2009IN VITRO-SELECTED Dap r STRAINS OF S.AUREUS 2315on June 26, 2014 by guest/Downloaded fromaction of many CAPs is the relative state of CM order (ﬂuid-ity).Thus,intact CMs or artiﬁcial membrane vesicles or bilay-ers,which are either very ﬂuid or very rigid,tend to be resistant to CAP-induced permeabilization (45).This has led to the notion that for each speciﬁc CAP-CM interaction,there is an optimum relative CM order at which individual CAPs exert maximal activity.For example,we previously showed that the CMs of S.aureus strains exhibiting in vitro resistance to the platelet CAP tPMP-1demonstrated signiﬁcantly more ﬂuid CMs than tPMP-1-susceptible strains (29).Alternatively,ren-dering such strains as CW-free protoplasts by enzymatic (lyso-staphin)CW degradation induced a shift in the CM order toward very rigid CMs,which also was associated with tPMP-1resistance (45).In our prior investigation of clinically derived Dap r strains (12),such organisms tended to have more ﬂuid CMs than the original parental isolate (12).In contrast,in the current study,as in vitro-passaged organisms acquired increas-ingly Dap r phenotypes,their CMs became more rigid.This observation raises a number of possibilities:(i)alterations in CM ﬂuidity are a nonspeciﬁc response mechanism in S.aureus to many different CAP exposures and are not precisely linked to the CAP-R phenotype;(ii)different CAPs have distinct structure-function relationships that could dictate the nature and scope of CM ﬂuidity shifts associated with peptide resis-tances;and (iii)in vitro-derived Dap r strains are exposed only to a single CAP,while clinically derived Dap r strains have been exposed to any number of innate host defense molecules dur-ing disseminated endovascular infections (including CAPs such as those from platelets and polymorphonuclear leukocytes)(12,25).This concept is supported by our recent documenta-tion of cross-resistance in clinical Dap r strains to both tPMP-1and hNP-1(a prototypic polymorphonuclear leukocyte CAP)(12).It is of interest that despite the lack of exposure to host defense CAPs,the Dap r laboratory strain in this study did show reduced killing by the two prototypical CAPs described above.This result suggested that CM perturbations induced by in vitro passage in DAP evoke a generalizable adaptation to un-related CAPs.The exact mechanism of altered CM ﬂuidity in Dap r strains is not well understood,but it does not appear to be linked to shifts in fatty acid unsaturation indices or branched chain species (18).Another notable feature of clinical Dap r strains character-ized in our prior investigations was an increased positive sur-face charge compared to those of their isogenic parenteral strains (12).Since the mprF and dlt operons are intimately involved in maintaining positive surface charge in S.aureus ,it follows that mutations in these loci could substantially impact the net surface charge.In our studies of Dap r clinical strains,we found an association between mprF point mutations and a gain-in-function phenotype (12).This was manifested by the increased translocation of the positively charged PL species LPG to the outer CM leaﬂet.Gain-in-function MprF alleles in Dap r clinical strains also have been seen in other studies (9,11,22,24,41).This underscored the potential for the charge-mediated surface repulsion of DAP as one explanation for the Dap r phenotype.In this context,the present investigation con-ﬁrmed a gain-in-function MprF allele (associated with both increased synthesis and the translocation of LPG in more pro-gressively Dap r strains)as well as enhanced dlt operon expres-sion during serial DAP passage.Whether these enhanced mprF and dlt operon expression proﬁles are directly related to SNPs or involve other regulatory loci such as aps/graRS is not known (6,42).Interestingly,in the present study,notwith-standing the increased expression proﬁles of dlt and mprF and phenotypic gains in function,the net surface charge paradox-ically became less positive during in vitro Dap r selection.This suggested the intriguing concept that other surface factors signiﬁcantly contribute to the net envelope charge.The prin-cipal candidate in this regard would be the extent of CW glutamate amidation,which previously has been linked to gly-copeptide resistance in MRSA VISA strains (22).The obser-vation of thickened CWs among the Dap r isolates in this in-vestigation supports such a paradigm.Thus,taken together,the enhanced negative surface charge in the in vitro-selected Dap r strains could conceivably facilitate rapid early DAP sur-face binding,while the speciﬁc upregulation of mprF func-tion and/or the increased CM rigidity in these strains sub-sequently could elicit a CM-speciﬁc DAP repulsion.Such a sequence of events then would collectively read out as re-duced DAP binding.It is important to point out that increased CW thickness has been associated with both the Dap r and VISA phenotype in two studies (4,5).Although postulated to be an afﬁnity-trap-ping mechanism for Dap,as documented for vancomycin in VISA isolates (5),this mechanism has not been conﬁrmed to date.Moreover,as for VISA strains,the thickened CW phe-notype also may be related to altered CW synthesis or per-turbed autolytic pathways (5).Further,it also is possible that the CW thickening is merely a nonspeciﬁc secondary adaptive response to a primary alteration in CM ﬂuidity or function (5,15).Of note,we demonstrated thickened CWs in our Dap r mutant in association with the VISA phenotype by both MIC testing and population analysis,despite the organism never being exposed to vancomycin.This same phenomenon wasTABLE 3.PL composition and asymmetry of LPG for the study strains aStrain%of total PL content (mean ϮSD)Inner b LPGOuter b LPGTotal LPGPGCLCB111811.16؎3.5 1.21؎0.6112.37؎3.383.96؎0.67 5.38Ϯ3.6CB220110.92Ϯ2.2 1.37Ϯ0.2612.29Ϯ2.086.37Ϯ3.3 3.60Ϯ0.0CB220211.64Ϯ2.4 1.70Ϯ0.6513.34Ϯ2.180.95Ϯ4.38.59Ϯ3.3CB220315.41Ϯ2.5 2.56Ϯ1.517.96Ϯ1.172.93Ϯ6.812.70Ϯ1.8CB220518.61؎4.4 6.00؎2.524.61؎3.570.02؎9.17.43Ϯ7.8a Substantive differences between the parental strain and the 20-day DAP-nonsusceptible passage mutant are in boldface.bMembrane leaﬂet.2316MISHRA ET AL.A NTIMICROB .A GENTS C HEMOTHER .on June 26, 2014 by guest/Downloaded from。