Membranes constitute the interface between the basic unit of life—a single cell—and the outside environment and thus in many ways comprise the ultimate “functional biomaterial”. To perform the many and often conflicting functions required in this role, for example to partition intracellular contents from the outside environment while maintaining rapid intake of nutrients and efflux of waste products, biological membranes have evolved tremendous complexity and versatility. This article describes how membranes, mainly in the context of living cells, are increasingly being manipulated for practical purposes with drug discovery, biofuels, and biosensors providing specific, illustrative examples. Attention is also given to biology-inspired, but completely synthetic, membrane-based technologies that are being enabled by emerging methods such as bio-3D printers. The diverse set of applications covered in this article are intended to illustrate how these versatile technologies—as they rapidly mature—hold tremendous promise to benefit human health in numerous ways ranging from the development of new medicines to sensitive and cost-effective environmental monitoring for pathogens and pollutants to replacing hydrocarbon-based fossil fuels. For example, according to one recent market analysis [The interface between the cell membrane and the extracellular matrix is a critical barrier that signaling molecules, other cells, pathogens, and environmental cues must traverse to modify cellular physiology. The membrane bilayer, however, is not just an obstruction but has evolved to be an exquisite enabling mechanism for signal transduction that contains a myriad of inserted transmembrane and carrier proteins that act as channels, transporters and signal transduction mediators that serve critical functional roles in adhesion, cellular communication, and bridging the extracellular environment with the cellular cytoskeleton. These factors position membranes as prime engineering targets to develop platform technologies ranging from whole cells to synthetic constructs based on concepts derived from cells. Applications for these technologies range from research tools that facilitate drug discovery and help understand complex cell-cell interactions to the creation of non-natural transmembrane protein systems for purposes including energy production, biosensing technology, and mitigation of environmental pollutants. Many analysis , the gloin vitro setting.In this article, we outline emerging strategies for engineering the cellular membrane by first providing an overview of technologies that utilize living cells and then by introducing cell-free systems inspired by nature in . Next, iCell-based membrane platforms are actively being developed for purposes ranging from biosensing, biofuel synthesis, and drug screening and technologies used to achieve these goals include installing a protein or system of proteins into the lipid bilayers of a cell through genetic manipulation, microinsertion, or by engineering the biogenesis of membranes by including lipid production as a design parameter. Although cell-based platforms offer important advantages such as fast response times in a physiologically relevant setting and naturally contain much of the machinery needed for recombinant protein synthesis they also have drawbacks. For example, they often suffer from a limited shelf life, a strict requirement for aseptic techniques, and the time investment needed to grow cells. In order to address these issues, cell-free synthetic systems pursue many of the same goals while avoiding the complications arising for the complexity of living systems. Accordingly, to provide perspective on both approaches, in this article we highlight a sampling of emerging technologies to illustrate both cell-based and cell-free platforms.Living cells encompass an elegant, sophisticated, and versatile platform for membrane-based technologies and attempts are underway to utilize them for applications ranging from drug development, protein over-expression, biosensing, to pollution mitigation. These applications typically require the biomolecular engineering of transmembrane proteins, which remains difficult to achieve in many cases and to further complicate matters, a lipid bilayer must be embedded with proteins that are properly folded and correctly oriented to create a fully functional biological membrane. In mammalian cells, membrane proteins depend on an extraordinarily complex quality control system during manufacture that has at its core the molecular chaperone proteins calnexin and calreticulin that ensure correct folding; this system also critically depends on N-glycosylation ,3. BecauIn some cases, increased expression of transmembrane proteins already present in a cell is the desired membrane engineering endpoint but more commonly the design goal is to insert proteins that are not normally found in host cell. For example, most systems designed to be biosensors or for the mitigation of environmental pollutants require the introduction of membrane proteins—or in some cases an entire system of transmembrane proteins—not normally found in the host cell. In either case, there is a requirement to not only over-produce properly folded and biologically active proteins but to also manipulate the cellular machinery responsible for membrane biosynthesis because insufficient lipid bilayer biogenesis can profoundly limit the amount of protein ultimately produced. Another problem that often arises is that particular classes of proteins including membrane transporters, porins, and channel-forming proteins that control membrane permeability and affect delicate intracellular chemistry tend to be toxic to host cells . ConsequThe engineering of membrane biology in cell-based systems is yielding insights that are aiding in the design of manufactured platforms that strive to mimic cellular biology through entirely synthetic “artificial membrane” systems. Advantages of synthetic platforms include the potential for higher throughput, greater stability from degradation for both reagents and products, and simplified experimental systems that result from avoiding competing reactions already present in a cell’s metabolic networks. Examples of promising advances resulting from early stage implementation of cell-free synthetic platforms include MembraneMax™ protein expression kits that utilize nanodisc biolayer and Arrowhead Research Corporation’s successful phase I clinical trial of CALAA-01, a polymersome that delivers patented siRNA against the M2 subunit of ribonucleotide reductase (RRM2) by inserting transferrin into the polymersome to target transferring receptors that are often over-expressed in cancer . One disBased on the promise of synthetic, cell-free membranes, there have been recent attempts to fabricate artificial systems using nanostructures that mimic the physiological properties of natural lipid bilayer membranes fused with transmembrane proteins. As discussed in a comprehensive review , temperaMembrane-based technologies are critical in biomedicine—in particular in drug development and production—in several ways. An overview of this topic is given here in in vitro tools in preclinical work are desperately needed to identify and guide early stage drug candidates through development with improved odds of translatable success into the clinic. Because of abysmal failure rates of lead candidates during clinical testing reasonably reflects back to insufficient models for testing drugs at early stages of development, much effort is devoted to elucidating the mechanisms by which novel drug molecules act, understanding the biochemical pathways they impact, and the cell wide molecular interactions they make because these factors are crucial for predicting efficacy and the potential for adverse effects for new drug candidates [Early stages of drug development, an integral aspect of the pharmaceutical industry, currently has a poor record of predicting therapeutic candidates that will ultimate experience clinical success; for example phase II testing eliminated ~82% of compounds as viable drug candidates . Clearlyndidates . The relhttp://zhanglab.ccmb.med.umich.edu/GPCRSD/) despite their incredible importance as drug targets. In general, even today obtaining detailed biochemical information through structural characterization of membrane bound proteins ranges from being “quite tedious” to being completely intractable due to the technical difficulties associated with obtaining sufficient quantities of properly folded membrane proteins for crystal structure determination. Isolation of membrane proteins involves extraction with detergents, refolding with detergent , and inDespite numerous advances in molecular biology technologies over the past few decades, the overexpression and structural characterization of membrane proteins remains challenging, thereby hindering the tremendous potential of these methods to guide drug discovery. An example of such a benefit lies in estimates that structure-based drug design (SBDD) can reduce costs of drug discovery from target identification to investigational new drug (IND) filing by almost 50% largely due to the improved quality of lead candidates that results from high quality structural information ,15. One Escherichia coli) expression systems have long been used to generate water soluble, usually cytosolic, recombinant proteins [E. coli cannot accommodate without further engineering. Post-translational modifications such as phosphorylation are widely recognized to control protein function and require no further discussion here. The role of glycosylation is often not fully appreciated but cannot be overlooked because it is critical to properly fold a protein for compatibility with the lipophilic environment of the cell membrane [One of the first bottlenecks that must be overcome to tackle a structure-function protein biology problem—for example, to determine how a drug candidate interacts with a GPCRs—is obtaining the hundreds-of-milligram quantities of protein required for X-ray crystallography. While bacterial  cells ,29, prodEfforts are underway to understand membrane biogenesis in more depth to learn how to manipulate the eukaryotic endomembrane synthetic machinery to increase surface area so as to accommodate accumulating integral membrane protein levels in engineered expression systems ,32,33,34PAH1, the gene that encodes phosphatidic acid phosphatase (Pah1p), acts as a gate keeper for partitioning flux of fatty acids into storage as TAG [PAH1 is deleted, diacylglycerol (DAG), and subsequently TAG, biosynthesis is disabled and metabolic flux in a cell is redistributed to produce excess membranes (Fatty acids are absorbed from culture media by eukaryotic cells and then stored in lipid droplets in the cytoplasm as triacylglycerols (TAG) and steryl esters (SE) . If store as TAG . This geembranes  that facYarrowia lipolytica—a ‘‘non-conventional’’ yeast capable of producing important metabolites and having an intense secretory activity, for use in industry as a biocatalyst, in molecular biology, and in genetics studies [PAH1 deleted genes (Δpah1) [Y. lipolytica wild type and Δpah1 strains had similar proliferative capacities, indicating that deletion of PAH1 has no serious impact on viability that would preclude the knockout strains from being used as hosts for producing recombinant membrane proteins. Next, the adenosine A2A receptor (A2AR), a member of the GPCR superfamily, was expressed by integrating the A2AR gene under the oleic acid-inducible POX2 promoter in both wild type and Δpah1 strains of Y. lipolytica. Furthermore, to increase the capacity of Y. lipolytica for protein production, the endoplasmic reticulum quality control mechanism for unfolded proteins, also known as the unfolded protein response (UPR), was concomitantly engineered by transforming both the wild type and Δpah1 strains with spliced active HAC1, a non-canonical intron mRNA that leads to translation of transcription factor Hac1p that activates the UPR, which was also driven by the POX2 promoter. Hac1p is found only in ER stressed yeast that has accumulating unfolded protein in its endoplasmic reticulum [pah1 strains was to fully integrate strains of yeast with increased membrane biogenesis with other metabolic engineering targets that have been identified as critical control mechanisms to regulate the synthesis of properly folded proteins.The regulation of genes involve in fatty acid metabolism is well established making it possible to engineer  studies  for incr (Δpah1) . Growth eticulum , thus thpah1 mutation profoundly enhanced production of over-expressed recombinant transmembrane protein A2AR [2AR production was due to not stronger promoter activation; rather, the driving force that led to higher quantities of A2AR was the deletion of PAH1 and the simultaneous over-expression of HAC1; the Δpah1/Hac1p combination resulted in the production of large quantities of fully functional A2AR through enhanced ER quality control coupled with increased membrane synthesis. Increased membrane synthesis was rigorously documented by electron microscopy, which showed that the Δpah1 mutant strains produced an increased amount of intracellular membranes compared to that wild type strain. The wild type strain produced massive quantities of TAG that were observed as intracellular lipid droplets when the yeast was grown on oleic acid. In contrast, the Δpah1 mutant strains were almost completely devoid of droplets in agreement with the hypothesis that enhanced lipid flux within the Δpah1 mutated strains was diverted towards membrane biogenesis. Finally, as a demonstration that these membrane engineering techniques were broadly applicable, the production of additional membrane proteins  NS4, presenilin/SPP homolog of Methanoculleus marisnigri, and human cytochrome P450 2D6) were evaluated in Δpah1 mutant strains and all showed enhanced expression [Western blot analysis showed that the Δein A2AR . Furtherpression .in vitro tools and reagents to aid drug screening and capture of transmembrane proteins. The need for these technologies is illustrated by GPCRs; biologically they gain their role as a prevalent drug because they initiate signaling cascades [in vitro testing platform that enables (amongst other capabilities) screening of mutations associated with disease.As discussed above, there are many complications and experimental challenges associated with solving crystal structures for GPCRs and other cell surface proteins. However, overcoming one of the first bottlenecks—the task of simply obtaining a sufficient quantity of high quality cell surface protein—may soon be solvable through cleverly designed cell membrane engineering strategies that increase membrane surface area sizes coupled to endogenous protein folding correction machinery as discussed above. Once a facile production system is in hand, additional opportunities arise for the study and exploitation of GPCRs and membrane proteins in general. For example as described here in cascades  implicatcascades . While ecascades , a speciPolymersomes are cell-sized artificial vesicles typically having a diameter of 100 nm to 10 μm that usually contain an aqueous solution in their core laden with biological molecules such as drugs, enzymes, or nucleic acids. Polymersomes can be thought of as “second generation” liposomes that have overcome limitations such as fragility, sensitivity to oxidation, and instability due to the inherent labile nature of lipids and lipid constructs by incorporating synthetic block copolymers into vesicle design. Polymersomes are generally designed to be encapsulated containers for delivery of sensitive biological agents or to serve as internal reaction chambers cordoned off from the surroundings by the membrane. Increasingly, they also offer a promising avenue for the display and production of membrane proteins; to date, several examples exist where membrane proteins have been introduced into a polymersome bilayer ,43,44. Pblock-poly(lactic acid) (PEG-b-PLA); the PLA homopolymer was added to increase the hydrophobicity of the polymersome membrane. By using this strategy, a proportion of MreB synthesized within the polymersome migrated and anchored into the polymer bilayer despite the absence of the elegant secretory apparatus found in eukaryotic cells due to interaction between a hydrophobic helical domain within the MreB protein and hydrophobic polymer membrane. In a second example [A recent approach  for synt example , polybut example . By inclWhile this early proof-of-principle example provides evidence that polymersomes can serve as vehicles for display of membrane proteins as “proteopolymersomes,” several questions remain including whether the proteins that insert into the polymersome membrane are correctly folded , biologically active, and consistently oriented inwards or outwards as desired. Additionally, several challenges remain for reproducible, robust, and scalable synthesis of polymersomes. Cell-free membrane expression systems that utilize a polymersome platform as cell-membrane mimetics hold promise as they can potentially circumvent the difficult problems that inevitably arise with overexpressing integral membrane proteins in cellular systems such as incorrect folding, cytotoxicity, and protein aggregation. Polymersomes can also avoid the labile nature of cell-free expression systems that use liposomes or other lipid based structures to create other artificial bilayer structures. Artificial bilayer structures and synthetic membranes are faster and a more economical alternative to animal or cell-based testing. Moreover, the use of synthetic membranes allows for large scale industrial testing capabilities, which accelerates the drug discovery process. Examples of institutions researching synthetic membranes include Agency for Science, Technology and Research’s (A*STAR) Artificial Cell Membranes (ACMs) Biolabs and the U.S. Department of Energy’s Argonne National Laboratory. Generating synthetic membranes with functional membrane proteins can thus create nearly identical, chemically controlled environments in which the activity of drugs can be tested. Synthesis of the artificial membranes involves mainly polymeric materials together with membrane proteins that are embedded in the nanosphere structure. Because target proteins are inserted into artificial membranes in isolation, prevention of additional background noise related to non-specific binding of drug molecules is achieved, resulting in lower interference signals in binding and transport assays.One novel approach to present functional membrane proteins in a defined and easy-to-study manner has involved the development of nano-sized lipid bilayer discs referred to as nanodiscs or nanolipoprotein particles (NLPs) ,47,48,49in situ [Briefly, NLPs can be synthesized by utilizing amphipathic α-helical proteins that can self-assemble into bilayer disc-like structures with lipids, where the edge of the discs are defined by the α-helical proteins that stabilize the leaflets ,52,53,54in situ . Finallyin situ ,58,59,60in situ ,62,63 itBased on the vast amounts of solar energy available , however, lead the overall energy conversion efficiency to be much lower ,68. As aChlamydomonas reinhardtii that were engineered with RNAi technology to permanently down-regulate LHC proteins, leading to a strain with much lower photoinhibition and greater biomass production [A specific example of this paradigm is provided by the Light Harvesting Complex (LHC) proteins involved in photosynthesis. LHCs are thylakoid membrane-bound proteins that bind pigments such as chlorophyll and carotenoids and orient them in an optimal configuration for photon capture. LHC proteins are finely regulated from level of mRNA transcription to protein degradation ,73,74,75oduction ,82.Once primary biology-based concerns in biofuels production such as inefficiencies arising from protective LHC mechanisms have been addressed, a second and often even more problematic challenge is physically obtaining the product from the host cell. Indeed, one of the most expensive steps in the downstream processing of biofuels is the harvesting and extraction of the product from biofuel-synthesizing cells. Consequently, intense research efforts focus on altering cellular membranes and membrane-bound transporters to facilitate the secretion of biofuels . UnfortuRather than produce biofuels within a cell, which involves facing the challenges associated with the extraction of the synthesized product, another option is to produce the product outside of a cell by having the requisite enzymes secreted from a cell or embedded into the plasma membrane such that their catalytic domains are displayed on the cell surface. This strategy not only reduces the costs involved with extraction of a biofuel from a host cell and subsequent purification from cellular debris, but also bypasses limitations in substrate uptake. Limitations in substrate uptake are especially acute in situations where the cell used in the energy extraction process is not actually the primary organism that harvested the solar energy; an illustration is a corn plant that uses photosynthesis to produce complex lignins, cellulose, hemicellulose and other forms of complex biomasses. A major challenge is that most “raw substrates” such as these are large insoluble materials that cannot be taken up and utilized by the micro-organisms that process them into useable biofuels and the process of converting bulk biomass into nutrients appropriate for microorganism processing represents one of the highest energy costs of the biofuel process .S. cerevisiae were engineered to co-display glucoamylase and α-amylase, thus allowing the yeast cells to process starch as a substrate [To improve biofuel production from “raw” plant matter, an attractive solution is to engineer cells that display enzymes necessary for the breakdown of complex biomacromolecules on their membranes with the catalytic domains oriented into the extracellular space. Cell surface technologies suitable for this purpose were initially developed for antigen presentation to facilitate vaccine production ,94, but ubstrate ,97,98. Uubstrate . Other yubstrate . Anotherubstrate ) and ferubstrate ,102.Biofuel cells are energy devices inspired by nature in their use of naturally occurring food sources as fuel but differ from the systems discussed above insofar as their goal is to produce electricity rather than chemicals or liquid fuels. In addition, although “biofuel cells” typically use enzymes in this process, they can also incorporate living, biological cells  into their design; in either case electricity is produced by oxidizing a variety of organic substrates. Because these systems can be designed to consume organic components of waste materials, they hold promise as an attractive way to generate an environmentally friendly power . HoweverArtificial photosynthesis attempts to replicate the natural process of photosynthesis that converts sunlight, water, and carbon dioxide into carbohydrates and oxygen . The terBiosensing, in the most rudimentary sense, describes technology that contains a biological component used for the measurement of a biological or chemical signal. Cell-based biosensors offer a rapid, reliable and cheap method to simultaneously test for multiple toxins that pose a threat to human cells. The specificity of cell-based biosensors has been largely dependent on the use of surface receptors on cell membranes to measure secondary responses to ligand molecules . AccordiA fundamental requirement for using cells as biosensors is an ability to convert a chemical, biochemical, or biological signal into a “physical” signal such as an electrical signal or a light-based readout. A novel biosensory method was reported in 2001 for detecting chemical and biological molecules by monitoring the electrophysiological interactions of the compounds with immobilized cells. This method was capable of detecting a range of targets including hepatitis C virus in human blood, plant viruses, and the herbicide glyphosate in aqueous solutions rapidly (in 3–5 min) and sensitively (at <100 pg/mL) . This tyE. coli [E. coli. One use for these biosensors is for the detection of pathogens in food samples [2–104 colony forming units per gram (CFU/g) and toxins in amounts as low as 10–40 ng.The ability to modify a cell surface with pathogen-specific antibodies allows mammalian cells to act as biosensors for the detection of a variety of viruses and pathogens. In addition to BERA, another biosensor approach that relies on immunoglobulin specificity is the Cellular Analysis and Notification of Antigen Risk and Yields (CANARY) sensor . This meE. coli ; subsequ samples  such as The nervous systems of higher animals, which consist of a complex balance of ionic fluxes and electrical impulses transmitted through an intricate network of neuronal cells, have inspired the pursuit of neuronal-based electric biosensors that have extraordinary sensitivity towards external chemical presences. These sensors utilize functional assays that couple the excitable property of neurons with computational genomics as a means to analyze neuronal exposure to environmental toxins ,120. Speet al., formed neuronal networks from neural stem cells that gave rise to multitudes of synaptically interlinked neurons with supporting astrocytes [A agonists as well as various other inhibitors, thereby indicating the presence of functional synaptic connections. Importantly, these neuronal networks that were grown from neural stem cells functioned similarly to those derived from primary neurons, thus providing a foundation for translation into use for biosensors. Once basic techniques are in place to construct neuronal networks, increased insight into their function is needed; one way to achieve this objective is through in silico modeling techniques that combine stochastic ion channel models with whole-network models that predict the bioelectrical response of neuronal networks [For sensomines, or similar cell-based sensing networks, to become robust, easy-to-deploy biosensors, several practical hurdles must be overcome. For example, reproducible and low cost methods to seed neurons onto microelectrode arrays and have them grow into functional neuronal networks are needed. In one strategy O’Shaughnessy trocytes . The netnetworks . SpecifiEnvironmental monitoring, which is becoming an increasingly important economic sector, requires quick and precise tools. For example, the detection of toxins and pathogens in food products as well as in water supplies is an urgent—and in many cases still unmet—public health concern. Traditional methods of pathogen and toxin detection and identification for these purposes rely on low throughput and cumbersome techniques such as the polymerized chain reaction (PCR) and culture techniques that often require counting colonies. Environmental monitoring has also been carried out by plant and animal-based assays utilizing algae, plants, as well as fish. However, many times these assays can be expensive and time consuming making them implausible to run on sufficiently large scales often needed for consistent and widespread environmental monitoring. In general, it is crucial that detection assays are sensitive to even the smallest concentrations of harmful compounds or pollutants, can be completed rapidly, and are cost-effective. The shortcomings of current efforts to detect pathogens, toxins, and pollutants in these regards have spurred increasing efforts towards using biosensing technology for higher throughput, simplicity of detection, and fast response times. Bioluminescent bacteria used as biosensors have favorable speed and cost considerations and also compare favorably to more traditional assays that employ algae and fish . SimilarAn attractive means of monitoring environmental pollutants, especially fresh water streams, is through the measurement of the five day biochemical oxygen demand (BOD). BOD sensors were first developed over 40 years ago but the sensors themselves remain unstable and the measurements are often difficult to reproduce . The senSimilar to many of the other emerging membrane engineering applications outlined in this article, cell-free strategies have distinct advantages over cell-based systems for biosensor development. In particular, conversion of this technology to a microarray format promises unprecedented opportunities for rapid and high-throughput detection and sensing platforms. In the past few years, several efforts have been reported towards this goal. In one example, supported lipid bilayers (SLBs) have been deployed in microarray format by first converting the SLBs into spherical supported lipid bilayers (SSLBs) on submicron-sized silicon dioxide beads . Beads cThe development of automated microfluidic methods to create planar membrane assemblages with embedded proteins  is openiRecent advances in three dimensional printing techniques have made it possible to design increasing complex biomaterials for use in cell and tissue engineering. Three dimensional printing began as a way to manufacture simple 3-D geometrical shapes but is now capable of implementing complex and elaborate network designs. Bio 3-D printing refers to the use of this technology to produce biological constructs ranging from bones and artificial skin to biomimetic membranes, which is of much interest to the field of cell and tissue engineering because this methods provides a new frontier for the development of tissue and customized cell membranes. Bio 3-D printing allows the creation of three-dimensional geometric structures made from aqueous solution droplets joined together by single lipid bilayers such as liposomes, which are vesicle-like droplets surrounded by a lipid monolayer approximately one picoliter in size. The liposomes are printed in layers on top adjoining their monolayers to form a lipid bilayer between them. Their lipid bilayers can be functionalized into highly engineered 3-D scaffolds by adding membrane proteins, and establishing electrical communication across the membranes. Networks can consist of tens of thousands of liposomes with at least several thousand layers, thus forming a tissue-like printed material.A basic 3-D bio printer consists of two glass capillary nozzles with thin needle tips with each joined to a chamber filled with an aqueous solution and capable of providing droplets of different aqueous compositions. Computer-controlled electronics modulate voltage to a piezoelectric transducer located in the back of the nozzles to administer the droplets . The priIf a tissue is to survive and adapt to its environment, the cells in the tissue need to have open communication with the environment and be able to transport molecules through their membrane compartments when necessary. Exchange of materials across the membrane and electrical communication are very important for the functionality of biomimetic membranes. Printing stable networks that mimic real tissue functions can be achieved through the insertion of membrane proteins in printed lipid bilayers and osmotic water flows can be manipulated by varying salt concentrations. Membrane conductivity has been achieved through the insertion of wild-type Staphylococcal α-haemolysin (αHL) into the droplets of aqueous solution ; αHL is Osmosis is the movement of water from a region of low solute concentration (high water concentration) to a region of high solute concentration (low water concentration) and osmotic balance must be maintained to print a stable and useful biomimetic membrane. Living cells use several different methods to avoid swelling due to osmosis, for example they contain water pores and channels that regulate osmotic balance and thus prevent swelling or shriveling. Biomimetic membranes also have to be able to maintain osmotic balance and once again, transmembrane proteins are used to control osmotic flows by transporting solute in and out of the cell, and thus, maintain osmotic balance.Osmosis can also be manipulated as an important tool in engineering droplet networks to attain certain geometries that can be difficult to print directly. The lipid layer of each droplet surrounds a certain amount of aqueous solution which can be tailored to specific printing needs. A higher concentration of salt in one droplet than in the surrounding droplets will cause it to swell with water while the surrounding cells shrink accordingly until the salt concentration is the same for all droplets in the network. The osmotic flows of water across the bilayers of the droplets will alter the conformation of the network through self-folding. For example, a planar flower shaped membrane with four petals can be printed in which the lower layers have lower osmotic gradients than the upper layers. This will cause the lower droplets to swell causing the network to fold inward and closing the flower creating a stable and self-supporting hollow sphere.sn-glycero-3-phosphoethanolamine (DOPE), and a lipid that stabilized the bilayer at a pH above its pKa, such as oleic acid [a of the oleic acid but destabilizes at lower pH due to the protonation of the oleic acid. When the pH becomes lower than the pKa the lipid bilayers break apart, releasing the internal droplet contents and mixing them with the external solution. The release of the contents across networks of droplets can be synchronized, with the simultaneous release of contents occurring within a minute [If osmotic flows could made reversible and controllable, a network of droplets could be printed that would fold and contract similar to other tissues in the body. A way to pursue this objective is through printed biomimetic membranes designed to display droplet sensitivity to pH and temperature changes. A step in this direction is by engineering the lipid bilayers of the droplets to release their contents at a certain pH . This fueic acid . Such a a minute . Dropleta minute . FinallyThere are many complicated factors affecting the function and conformation of networks of lipid droplets. Fortunately, predicting the final geometrical shape of the droplet network is possible through the use of computational models. Three-dimensional printer software is now able to determine the final geometry fairly accurately by taking into account the initial geometry, distribution of two types of droplets, and osmotic gradients. Through cell membrane engineering droplet networks have been given electrical communication by transmembrane proteins, such as αHL, and macroscopic folding due to osmotic flows. These properties are essential for manipulating biomimetic membranes and making them similar to real cells and tissue and increasingly, once a highly engineered membrane can be designed, three-dimensional printing can create it.One of the most important cellular architectures is the plasma membrane, which not houses an entire cell, but also provides a unique environment for transmembrane proteins. Although bioscience is becoming increasingly proficient at manipulating genomes and systems for protein production in many types of cells and in cell-free expression system, serious challenges remain for membrane proteins. For example, as discussed in Increasing success in these efforts is expected to pay off in a range of applications from drug development to biofuel production strategies . Insertiin vitro tools that in some cases utilize living cells and in other cases are completely synthetic. The new and exciting field of bio 3-D printing (in vitro tool stands to revolutionize our understanding of biology by allowing one to specifically manipulate cell mimicking structures in not only a 3-D environment, but also within a complex tissue-like environment, thus opening the door for advanced investigation of—and control over—cell-cell communication and behavior in various tissue architectures and environments.Despite the diversity of applications covered in this review, a common theme is the use of biological—or biology-inspired artificial—membranes in the design and assembly of printing  essentiain vitro tools and reagents based on engineered membrane systems already play important roles in the design, understanding, and discovery of medicines, biofuels production, and environmental monitoring, as outlined in this article.In the future, technology may advance to the point we will be able to construct functional cells through entirely synthetic means. Achieving this tremendous accomplishment will require additional advances in technology but also continued investigation of fundamental concepts, such as the fact that the cell membrane is one of the most important features of a cell that controls signaling cascades, temporal cues, cell-cell communication, cellular adhesion, and cell motility. At the present time,


PRNP) gene. However, the stability of transmissible spongiform encephalopathy phenotypes on passage across and within species is not absolute, and there are reports in the literature where experimental transmissions of particular isolates have resulted in a phenotype consistent with a different strain. In this study, intracerebral inoculation of atypical scrapie between two genotypes both associated with susceptibility to atypical forms of disease resulted in one sheep displaying an altered phenotype with clinical, pathological, biochemical and murine bioassay characteristics all consistent with the classical scrapie strain CH1641, and distinct from the atypical scrapie donor, while the second sheep did not succumb to challenge. One of two sheep orally challenged with the same inoculum developed atypical scrapie indistinguishable from the donor. This study adds to the range of transmissible spongiform encephalopathy phenotype changes that have been reported following various different experimental donor-recipient combinations. While these circumstances may not arise through natural exposure to disease in the field, there is the potential for iatrogenic exposure should current disease surveillance and feed controls be relaxed. Future sheep to sheep transmission of atypical scrapie might lead to instances of disease with an alternative phenotype and onward transmission potential which may have adverse implications for both public health and animal disease control policies.The interactions of host and infecting strain in ovine transmissible spongiform encephalopathies are known to be complex, and have a profound effect on the resulting phenotype of disease. In contrast to classical scrapie, the pathology in naturally-occurring cases of atypical scrapie appears more consistent, regardless of genotype, and is preserved on transmission within sheep homologous for the prion protein ( Sc of a host encoded cell membrane bound protein, designated PrPC. The occurrence of different prion ‘strains’ is inferred from consistent differences in disease phenotype—the observable clinical presentation, pathogenesis, pathology and PrP molecular characteristics of the diseased host—and the disease phenotype observed in wild type or PrP transgenic rodent models. The one factor which is consistent in all cases, and forms the principal component of the current statutory diagnostic methods for TSE, is the disease-specific accumulation of PrPSc as demonstrated by immunochemical techniques such as ELISA, immunohistochemistry or Western blotting.Scrapie belongs to a group of diseases known as the transmissible spongiform encephalopathies (TSEs) or prion diseases. Prions are believed to be composed exclusively of abnormal isoforms, designated PrPSmall ruminants are affected by at least two forms of naturally occurring scrapie which have different molecular and neuropathogical characteristics. Classical scrapie has been reported as a clinical entity in sheep for at least 250 years. Following experimental challenges distinct ‘strain’ characteristics can be identified , but whehttp://www.animalhealthaustralia.com.au/wp-content/uploads/2011/03/Australia-and-New-Zealand-Standard-Diagnostic-Protocols-for-TSE.pdf). In general atypical scrapie occurs as a single case on a farm, usually in older animals (over 5 years of age) and rarely presents with clinical signs that would be considered indicative of TSE [A distinctly different phenotype of ovine TSE, termed atypical scrapie or Nor98) has recently been recognised, although it is speculated to have existed undetected for much longer than this ,6. It is has recee of TSE . Atypicae of TSE ,14 and se of TSE  or the oe of TSE , with ree of TSE –20.PRNP gene confers susceptibility to classical and resistance to atypical scrapie; conversely, arginine (R) at codon 171 confers resistance to classical and susceptibility to atypical scrapie, and histidine (H) at 154 is associated with susceptibility to atypical scrapie [136R154Q171/ARQ sheep [There are remarkable similarities and differences between atypical and classical scrapie. Most notably, in sheep they both show affiliation to specific PrP genotypes, but breeding for resistance to classical scrapie enriches for genotypes that are susceptible to atypical scrapie and vice versa. As an example, valine (V) at codon 136 of the  scrapie ,22. TherRQ sheep . The comThe stability of TSE isolates on passage across and within species is not absolute, however, and there are increasing numbers of reports in the literature where experimental transmissions of particular isolates have resulted in a phenotype consistent with a different strain –27. IndeDespite naturally-occurring atypical scrapie being observed in a range of genotypes, successful experimental transmissions of clinical disease have so far only been reported within a particular homologous donor-recipient genotype model using sheep which are AHQ/AHQ homozygous ,15,16. TAll intracerebral inoculations were carried out under general anaesthesia, and in accordance with the United Kingdom (UK) Animal (Scientific Procedures) Act 1986, under Licence from the UK Government Home Office . Such licences are only granted following approval by the internal APHA ethical review process as mandated by the Home Office.The sheep described in this paper were challenged as part of a larger study that included in total seven donor and 54 recipient animals (excluding controls) to investigate the transmissibility and phenotypic stability of atypical scrapie in both homologous and heterologous donor/recipient genotype combinations. All recipient sheep were sourced from Defra’s New Zealand-derived flock, which is free from classical scrapie .Although this study is still ongoing emerging data have already been published with regard to successful homologous AHQ/AHQ transmissions ,15. DonoTwo VRQ/VRQ and two AHQ/AHQ sheep were challenged orally with 5g of neat brain homogenate . InoculuAll challenged animals were euthanized when they presented with clinical disease, or kept until seven years post challenge, as a pre-determined study endpoint. At post mortem, the whole brain was removed and hemisected longitudinally. One half of the brain was placed into 10% formal saline for histology, and the other half stored at -80°C. A range of lymphoid tissues were also collected.PRNP allele on a murine PRNP null background and are known to be susceptible to both atypical and classical scrapie [An aliquot of the same inoculum that was used for the intracerebral challenge of the two AHQ/AHQ sheep, the frontal cortex of the positive AHQ/AHQ recipient and frontal cortex from a CH1641 experimentally challenged sheep were screened for microbiological sterility prior to inoculation into mice. Panels of 10 tg338 transgenic mice which over-express an ovine VRQ  scrapie  were ino scrapie ,33.A neurological examination and behavioural observations were conducted when animal care staff suspected clinical disease .Sc was performed as previously described [154 R171–182, and specifically recognising the R151-R159 sequence. Tissues from the lymphoreticular system were immunolabelled using the same method. Brain immunolabelling was recorded and analysed using a ‘short PrPSc profiling protocol’ as described [Ovine brain tissue was routinely fixed, processed into paraffin wax, sectioned and stained with haematoxylin and eosin as described in detail elsewhere . Immunohescribed  using moescribed .Murine brains were sectioned parasagitally and the larger portion (approximately 2/3 of the brain) was fixed in 10% buffered formalin and processed for histological examination. Sections (3μm thick) were stained with haematoxylin and eosin, and the intensity of vacuolation was assessed semi-quantitatively at specific neuroanatomical areas and used to produce lesion profiles as described previously ,34.Sc was performed according to standard methodology using mouse monoclonal antibody R145 , which recognizes amino acids 221–233 of bovine PrP [Immunohistochemical detection of PrPvine PrP . The smaUnfixed brain samples were subjected to the TeSeE Universal WB  as previously described . The samFollowing initial observations that one challenged animal displayed unexpected characteristics which were similar to the classical scrapie strain CH1641 , unpubliThe two VRQ/VRQ sheep, and one of the AHQ/AHQ sheep, orally challenged with the ARR/ARR atypical inoculum were clinically normal at the end of the seven year challenge period, and were negative by all post mortem tests.During the pre-cull clinical examination at seven years post challenge, the other AHQ/AHQ sheep displayed mild ataxia, a wide-based hind gait and a fine head tremor. It was also teeth grinding. When blindfolded, it tended to circle to the left and displayed loss of balance. After the examination it aimlessly circled to the right in wide circles. It appeared to be confused and did not immediately follow other sheep . Three cSc in any lymphoid or peripheral nervous system tissues (data not shown).Pathologically, there was no detectable immunolabelling at the obex, and quite restricted labelling in the cerebellum and remaining brainstem. Cortical labelling was very diffuse, but widespread, as was the labeling within the basal ganglia. The clinical and pathological features of this animal, and the Western blot profile, were all consistent with atypical scrapie, including the absence of any detectable PrPOne sheep remained clinically normal until it was culled at seven years post challenge, and was TSE negative by all post mortem tests.The other sheep presented with acute onset clinical abnormalities at 514 days post inoculation (dpi). It was found in lateral recumbency by animal care staff who suspected an epileptic fit. Following this period of recumbency, the animal was reported to be circling or drifting to one side. These observations could not be confirmed during systematic neurological examination, which would be compatible with them being transient post-ictal signs, but the animal was found to have visual impairment  and hind limb ataxia (see movie PG 0306–08.mov). There was no wool loss suggestive of pruritus and it did not respond to scratching of the back (negative scratch test response). Subsequent CCTV observation indicated that the animal had difficulty lying down and standing up and was less active, standing occasionally in the pen with its head lowered, but no pruritic behavior was displayed. It was euthanized at 515 dpi.These clinical signs were similar to those exhibited by sheep challenged with experimental CH1641 (n = 4) which, had a mean incubation period of 463±2 dpi, displayed ataxia but had no evidence of pruritus and some also lacked a menace response . They were also similar to the most consistent clinical signs evident in AHQ/AHQ sheep intracerebrally inoculated with atypical scrapie brain from AHQ/AHQ sheep , although average incubation periods were longer and more variable range 378–1057), at 758±208 dpi (n = 9) [–1057, atSc short protocol mapping resulted in a profile that was indistinguishable from that of CH1641 [The immunolabelling seen throughout the neuraxis in this sheep was predominantly intracellular, in addition to the particulate labeling of the neuropil. . The PrPf CH1641  Fig. 2)Sc short f CH1641 .The Western blot profile obtained from this sheep exhibited a three band pattern with a low molecular mass migration, relative to classical scrapie, when detected with mAb SHA31. When detected with mAb P4, it also had a much reduced signal intensity compared to the classical scrapie control, which was similar to the reduction in signal observed in the ovine BSE and CH1641 positive controls. In contrast, the profile observed in the brain tissue from the donor corresponded to that of the atypical scrapie control, originally at a weaker intensity but clearly visible in the concentrated sample .Following deglycosylation and detection with mAb SAF84 the presence of a band at approximately 12kD was observed, the same as detected in the CH1641 control but not in the ovine BSE or bovine BSE control .Overall, the WB results characterized the donor as atypical scrapie and the recipient as CH1641-like scrapie.Sc types in the mouse brain was in agreement with the incubation period and lesion profile data . Additionally, an audit of all prepared CH1641 samples indicated that all the anticipated samples remain in our archive and could not, therefore, have been used in error.Another possible hypothesis is that the donor sheep might have been co-infected sub-clinically with a CH1641-like classical scrapie strain in addition to atypical scrapie. Such co-infection of different scrapie strains, while observed very rarely, has already been described in field situations ,37, and PRNP congenic TgBov mice [The third, and arguably the most plausible, explanation is that the original atypical isolate has ‘converted/mutated’ into CH1641 as a result of the experimental conditions. Again there is some precedent for this occurrence, with previous experimental observations in porcine transgenic mice, where an atypical scrapie isolate acquired a typical 3-band blot pattern with characteristics similar to sub-passaged ovine BSE . This exBov mice . If thisIt is likely that it will never be possible to determine conclusively what gave rise to this change in phenotype. However, this study adds to the growing body of data which highlights a fundamental problem with prion diseases; in the absence of an agent that is structurally independent form the host genome and which can be isolated and typed, classification of natural disease relies on the phenotypic characteristics of the host. Given that some isolates appear to be unstable under certain circumstances, there is a danger in making assumptions about the disease situation in the field, and the associated public and animal health risks based on observed phenotype alone.S1 FileA clinical examination carried out at 514 days post inoculation reveals a bilaterally absent menace response: the sheep does not blink in response to movement of a hand/finger towards the eye but blinks when the skin surrounding the eye is touched with artery forceps. Hind limb ataxia is present (excessive swaying of the hind limbs and awkward placement of the limbs when it moves in the corner of the corridor). It bumps its head against the corner of one of the food troughs protruding into the corridor when it turns and also moves once with its head very close to the food trough after it walks from the corner towards the camera, which suggests some visual impairment. It is however able to negotiate obstacles placed on the floor of the corridor without difficulty. Camera surveillance of the pen showed abnormal lying down and rising behaviour of this sheep (identifiable by the orange arrow) compared to its pen mates (identifiable by the white circle).(MOV)Click here for additional data file.


Recent research in identification of brain injury after trauma shows many possible blood biomarkers that may help identify the fetus and neonate with encephalopathy. Traumatic brain injury shares many common features with perinatal hypoxic-ischemic encephalopathy. Trauma has a hypoxic component, and one of the 1st physiologic consequences of moderate-severe traumatic brain injury is apnea. Trauma and hypoxia-ischemia initiate an excitotoxic cascade and free radical injury followed by the inflammatory cascade, producing injury in neurons, glial cells and white matter. Increased excitatory amino acids, lipid peroxidation products, and alteration in microRNAs and inflammatory markers are common to both traumatic brain injury and perinatal encephalopathy. The blood-brain barrier is disrupted in both leading to egress of substances normally only found in the central nervous system. Brain exosomes may represent ideal biomarker containers, as RNA and protein transported within the vesicles are protected from enzymatic degradation. Evaluation of fetal or neonatal brain derived exosomes that cross the blood-brain barrier and circulate peripherally has been referred to as the “liquid brain biopsy.” A multiplex of serum biomarkers could improve upon the current imprecise methods of identifying fetal and neonatal brain injury such as fetal heart rate abnormalities, meconium, cord gases at delivery, and Apgar scores. Quantitative biomarker measurements of perinatal brain injury and recovery could lead to operative delivery only in the presence of significant fetal risk, triage to appropriate therapy after birth and measure the effectiveness of treatment. Worldwide it is estimated that 1.15 million babies develop hypoxic-ischemic encephalopathy (HIE) every year  promotes brain damage through the release of free radicals, stimulating inflammatory reactions, and enhancing the toxicity of excitatory amino acids  are being investigated as promising serum biomarkers for neurotrauma. miRNA may be stable in the circulation which supports their potential use as disease biomarkers (Egea et al., A potentially transformative finding in traumatic brain injury research has been the identification of exosomes, which are nano-sized extracellular vesicles that have key roles in cell signaling and undergo membrane fusion so that they readily cross the blood-brain barrier. Exosomes contain proteins, miRNA and other nucleic acids, and are believed to play a major role in disposal of cellular waste (Werner and Stevens, Blood biomarkers discovered in traumatic brain injury could significantly improve the management of neonates with HIE, particularly those with mild and moderate injury, by providing more accurate early diagnosis and prognosis, and for monitoring therapies in the acute care setting (Papa et al., Serum based combined multi-marker analysis reflecting glial and neuronal cell damage should be considered for understanding more precise pathophysiological mechanisms of brain injury. Glial injury can be assessed by GFAP in the blood and axonal injury by C-Tau and spectrin protein breakdown products (Guingab-Cagmat et al., The pathology of perinatal HIE is very heterogeneous and one “magic” biomarker may not be the solution, but a panel of biomarkers may prove to be most useful in distinguishing the different pathologic-anatomic processes that comprise the injury (Papa et al., Blood biomarkers offer an objective and quantitative way to identify and follow a neonate with brain injury. They could be used to triage neonates to the current standard of 72 h of hypothermia as well as investigational therapies such as erythropoietin, xenon gas, melatonin and various forms of stem cell treatment. Blood biomarkers could possibly provide information about the extent of injury in the acute phase before ultrasound or MRI can identify abnormalities and determine the timing of injury as well. Blood biomarkers measured noninvasively using near-infrared spectroscopy (Torricelli et al., All authors listed, have made substantial, direct, and intellectual contribution to the work, and approved it for publication.Ernest Graham and Frances Northington are supported by the Cerebral Palsy International Research Foundation. Frances Northington is supported by R01 HD070996 and R01 HD074593. Irina Burd is supported by NICHD K08 HD073315-01. Allen Everett is supported by NHLBI R01 HL119664 and NIBIB R21EB018426The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer MO and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


Several interventions are available for management of obsessive-compulsive disorder in adults, but few studies have compared their relative efficacy in a single analysis. We aimed to simultaneously compare all available treatments using both direct and indirect data.In this systematic review and network meta-analysis, we searched the two controlled trials registers maintained by the Cochrane Collaboration Common Mental Disorders group for trials published up to Feb 16, 2016. We selected randomised controlled trials in which an active psychotherapeutic or pharmacological intervention had been used in adults with obsessive-compulsive disorder. We allowed all comorbidities except for schizophrenia or bipolar disorder. We excluded studies that focused exclusively on treatment-resistant patient populations defined within the same study. We extracted data from published reports. The primary outcome was symptom severity as measured by the Yale-Brown Obsessive Compulsive Scale. We report mean differences with 95% credible intervals compared with placebo. This study is registered with PROSPERO, number CRD42012002441.We identified 1480 articles in our search and included 53 articles  in the network meta-analysis. Behavioural therapy , cognitive therapy , behavioural therapy and clomipramine , cognitive behavioural therapy and fluvoxamine , cognitive behavioural therapy , clomipramine , and all SSRIs  had greater effects than did drug placebo. Clomipramine was not better than were SSRIs (−1·23 [–3·41 to 0·94]). Psychotherapeutic interventions had a greater effect than did medications, but a serious limitation was that most psychotherapeutic trials included patients who were taking stable doses of antidepressants .A range of interventions is effective in the management of obsessive-compulsive disorder, but considerable uncertainty and limitations exist regarding their relative efficacy. Taking all the evidence into account, the combination of psychotherapeutic and psychopharmacological interventions is likely to be more effective than are psychotherapeutic interventions alone, at least in severe obsessive-compulsive disorder.National Institute for Health Research. Obsessive compulsive disorder is considered the fourth most common mental disorder in high-income countries and ranks as the tenth leading cause of disability worldwide.Previous systematic reviews and meta-analyses have generally compared the efficacy of pharmacological interventions with placebo, not with each other.Evidence before this studyDuring the protocol stage of our project , we did a scoping search of the literature. We used the two specialised registers of controlled trials maintained and administered by the Cochrane Collaboration Common Mental Disorders Group. We searched the registers using the generic term “condition = obsess* OR compulsi*”, with no language or date restrictions. We found that the latest comprehensive review had been published in 2006 and specific meta-analyses had been published in 2008. Since then, several new trials have been done. Previous systematic reviews and meta-analyses have generally focused on the comparison between antidepressant medications and placebo or psychotherapeutic interventions and a waiting list. Few studies have directly compared the relative efficacy of serotonergic antidepressants versus each other, behavioural-type psychotherapies versus each other, or medications versus psychotherapies. Clinicians are often interested in pragmatic comparisons , but these questions have been examined in few studies in the past using statistical methods that have not always taken into account the complexity of such comparisons. We therefore did a network meta-analysis with the aim to simultaneously compare in a single analysis and rank in terms of efficacy all available interventions for management of obsessive-compulsive disorder in adults.Added value of this studyWe found small differences in efficacy between medications, and the hypothesis of clomipramine being better than SSRIs was not confirmed. Although certain psychotherapies were associated with larger effects than were medications, we underline an important limitation that, in most psychotherapeutic trials, patients who were taking stable doses of antidepressants were not excluded and therefore these therapies cannot be considered as pure monotherapies.Implications of all the available evidenceTaking all evidence into account, the combination of psychotherapies with medications is possibly the most effective intervention and clinicians should consider this option more often than at present for patients with severe obsessive-compulsive disorder. Psychotherapy is effective in symptomatic patients taking antidepressant medications, and its effect as monotherapy is not known. Future research should try to differentiate more clearly the effect of medications versus psychotherapy and monotherapy versus combined therapy, avoiding the limitations that we have underlined in this study.In this systematic review and network meta-analysis, we searched the two controlled trials registers maintained by the Cochrane Collaboration Common Mental Disorders group for trials published up to Feb 16, 2016, by experienced staff of the Cochrane Common Mental Disorders group using their standard methodology. Reports of trials for inclusion in the Group's registers are collated from routine (weekly), generic searches of MEDLINE, Embase, and PsycINFO; quarterly searches of the Cochrane Central Register of Controlled Trials; and review-specific searches of additional databases. We searched the registers using the generic term “condition = obsess* OR compulsi*”, with no language restrictions. We included studies in the review if they were randomised controlled trials of adult patients with a diagnosis of obsessive-compulsive disorder. We allowed all comorbidities except for schizophrenia or bipolar disorder. We excluded studies that focused exclusively on treatment-resistant patient populations defined within the same study.Eligible experimental interventions were all antidepressantsData extraction was done independently by two reviewers (HB and PSk) and validated by one reviewer (PSk). We used standardised data extraction Word forms and structured Excel spreadsheets to extract data from published reports. In cases of duplicate data, we selected the manuscript with the largest sample. We also considered preliminary congress abstracts duplicate and did not select them if a full article had been published after the congress. We extracted data for inclusion and exclusion criteria , general details of the study , details of continuous outcome assessment , and details of the risk of bias assessment .For the quantitative synthesis, the primary outcome measure was continuous and it was symptom severity as measured by YBOCS.We did pairwise and network meta-analyses for efficacy. We excluded studies that did not use YBOCS. This post-hoc decision was made for two reasons: YBOCS is the only available clinician-rated scale that has been extensively validated in controlled trials worldwideOpenBUGS version 3.2.3. We used the random-effect models described by Dias and colleagues,We did all analyses in a Bayesian framework using Preplanned sensitivity analyses excluded studies at high risk of bias for the following domains: masking of the outcome assessor, incomplete outcome data, and high overall attrition or evidence of differential attrition between groups. We present the results both before  and after excluding waiting list controlled trials. These trials are non-masked and evidence exists that they lead to biased results in favour of the active psychotherapeutic interventions.The funder had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.We identified 1480 articles in our search and assessed 158 (11%) full-text articles for eligibility . We exclThe 64 trials included in the qualitative review were published over a period of 33 years  involved 17 different treatments grouped into 12 classes  and those with psychotherapy arms only (18 [28%] of 64) are presented in the For the sensitivity analyses, we used the full network  or after exclusion of the waiting list controlled trials (for CBT), were more likely to lead to a larger effect than were medications. Some previous meta-analyses have reported similar results in favour of psychotherapy. For example, Cuijpers and colleaguesOur analysis has several limitations. Most studies were of short-term duration. As most of the studies that tested the efficacy of psychotherapeutic interventions included patients who were taking stable doses of antidepressant medications, generalisation of these results to patients not on medications is not possible. We were unable to test different doses of the same drug to investigate potential dose-response associations.The results of our analysis generally support current National Institute for Health and Care Excellence guidelines.Further research should try to differentiate more clearly than at present the effect of medications versus psychotherapy and monotherapy versus combined therapy. Trials that investigate the effect of psychotherapy should monitor use of antidepressants in included patients or recruit patients who are willing to taper off their antidepressant medication before entering randomisation. As obsessive-compulsive disorder is a very heterogeneous condition, more pragmatic trials of longer duration than have been done so far are needed to test the efficacy of existing interventions in patients encountered in daily clinical practice (including those with other comorbid conditions) and the augmenting effect of medications in addition to psychotherapy or vice versa in patients with treatment-refractory obsessive-compulsive disorder.


Diabetic nephropathy (DN) is one the most prevalent chronic complications of diabetes mellitus that affects as much as one-third of diabetic patients irrespective of the type of diabetes. Hyperglycemia is the key trigger for DN that initiates a number of microscopic and ultramicroscopic changes in kidney architecture. Microscopic changes include thickening of the glomerular basement membrane (GBM), tubular basement membrane (TBM), mesangial proliferation, arteriosclerosis, and glomerulotubular junction abnormalities (GTJA). Among the ultramicroscopic changes, effacement of podocytes and decrease in their density seem to be the centerpiece of DN pathogenesis. These changes in kidney architecture then produce functional deficits, such as microalbuminuria and decreased glomerular filtration rate (GFR). Among several mechanisms involved in inflicting damage to podocytes, injuries sustained by increased oxidative stress turns out to be the most important mechanism. Different variables that are included in increased production of reactive oxygen species (ROS) include a hyperglycemia-induced reduction in glutathione (GSH), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation via hyperglycemia, advanced glycation end products (AGEs), protein kinase C (PKC), and renin-angiotensin-aldosterone system (RAAS).Unfortunately, control of podocyte injury hasn’t received much attention as a treatment approach for DN. Therefore, this review article is mainly concerned with the exploration of various treatment options that might help in decreasing the podocyte injury, mainly by reducing the level of NADPH oxidase-mediated generation of ROS. This article concludes with a view that certain NADPH oxidase inhibitors, RAAS inhibitors, statins, antidiabetic drugs, and antioxidant vitamins might be useful in decreasing podocyte injury and resultant structural and functional kidney impairments in DN. Diabetes is a group of metabolic disorders that is characterized by persistent hyperglycemia either due to the destruction of beta pancreatic cells resulting in a deficit in insulin production or decreased responsiveness of body tissues to secreted insulin (or decreased insulin sensitivity), or both -2. As pePoor glycemic control in diabetes not only increases the risk of acute complications, like hypoglycemia and hyperglycemia , but is The gross microscopic picture of DN is characterized by increased thickening of glomerular basement membrane (GBM), which is perhaps the earliest detectable lesion in DN -14. As tDN in humans is characterized by microalbuminuria, which eventually can progress to proteinuria . ChangesHyperglycemia seems to be the centerpiece for podocyte injury and DN as a whole, which is most likely due to a higher degree of oxidative stress. Excess glucose is metabolized through multiple accessory pathways like the polyol pathway that converts glucose into sorbitol, which then depletes the amount of antioxidants like glutathione (GSH), and increases the level of reactive oxygen species (ROS) . In addiA significantly important limb responsible for oxidative stress-induced podocyte damage is the renin-angiotensin-aldosterone axis. Hyperglycemia causes a local increase in blood pressure due to the activation of the renin-angiotensin system (RAS) . In addiSo the question is how the generation of ROS causes damage to podocytes; ROS are well known for their potential to induce apoptosis in cells . NormallIn a crux, it is evident from the above discussion that hyperglycemia-induced oxidative stress is the key event in the injury inflicted to podocytes in DN. Therefore, improvement in our understanding of oxidative stress may prove to be the missing key in the pathogenesis of DN. Moreover, drug targets that can help improve the degree of podocyte survival by decreasing oxidative stress can prove to be valuable assets in treating diabetic patients with DN. Therefore, the purpose of this article is to find possible drug targets that have been shown to decrease ROS-induced podocyte injury and possibly improve DN status in patients suffering from diabetes mellitus. Podocyte injury has been implicated as one of the earliest events in DN. Injury to podocytes due to uncontrolled hyperglycemia and resultant podocyte apoptosis are the most significant events that seem to contribute in decreasing podocyte density, which can be evaluated as microalbuminuria . DiabeteAs discussed above, almost all hyperglycemia-activated pathways induce the production of ROS via the activation of NADPH oxidase. Thus, it can be safely speculated that inhibition of this enzyme may be the most effective therapy in reducing hyperglycemia-induced oxidative stress and resultant podocyte injury. In fact, this hypothesis has been backed by several studies where the use of different oxidase inhibitors has shown significant promise in putting a stop and rather reversing the oxidative stress-mediated podocyte injury. Triptolide is a known anti-inflammatory substance and is well-known for its ability to decrease oxidative stress. It brings about its action by causing inhibition of NADPH oxidase and inducible nitric oxide synthase (iNOS) . TreatmeAngiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor inhibitors (ARIs) have long been used for the treatment of both non-diabetic and diabetic kidney diseases. How these drugs help protect patients with kidney disease is still not completely understood and cannot be explained by mere regulation of blood pressure -64. EnalStatins are antihypercholesterolemic drugs that have beneficial antioxidant activity as well. The antioxidant ability of statins can be attributed to two of their properties. First, they decrease the activity of ROS-producing enzymes, such as NADPH oxidase -75. SecoPreferring some antidiabetic drugs over the others can be an interesting and useful treatment option for DN. This treatment option can have two-fold benefits as it only not provides better glycemic control but also provides renoprotection against oxidative stress-induced damage. The first antidiabetic drug that deserves to be mentioned is rapamycin. It has been shown to decrease oxidative stress and improve the function of antioxidant enzymes, such as glutathione -80. ReseAmong vitamins, the vitamins that might be useful in the treatment of DN include antioxidant vitamins like vitamins C, D, and E. Vitamin D is a well-known antioxidant  and inhiPodocyte injury represents a centripetal event in the progression of DN and related functional deficits like microalbuminuria and decreased GFR. NAPDH oxidase-mediated oxidative stress seems to be the most important event in the oxidative podocyte injury. Increased production of ROS then inflicts serious damage to the podocytes and leads to slowly progressing deterioration in kidney structure and function. Thus, blocking the production of ROS can be a valuable treatment approach for decreasing podocyte injury and resultant kidney disease. In the light of current literature, we have concluded that certain NADPH oxidase inhibitors, RAAS inhibitors, statins, antidiabetic drugs, and antioxidant vitamins have shown significant potential in decreasing oxidation-induced podocyte injury. These therapeutic agents have shown positive results in animal models of DN, but clinical trials to see their effects on humans are rather lacking. There is a need for research and clinical trials using these therapeutic agents to study their efficacy for managing DN.


Our conceptual and mechanistic understanding of how plant nucleotide-binding leucine-rich repeat (NLR or NB-LRR) proteins perceive pathogens continues to advance. NLRs are intracellular multidomain proteins that recognize pathogen-derived effectors either directly or indirectly (Jones and Dangl, In a recent article, Cesari et al.  elegantlHere, we expand on the Cesari et al.  model anHow to test whether or not SDs are decoys? We propose a straightforward genetic test that can reject the decoy hypothesis. Isogenic plants either carrying or lacking the NLR-SD can be challenged with a pathogen strain that lacks the matching avirulence effector Figure . There aOur goal is not to engage in an exercise in semantics. However, we wish to avoid conceptually restrictive terminology and urge the plant-microbe interactions community to test a rich spectrum of models and hypotheses. The proposed sensor domain terminology would accommodate this breadth of ideas. Ultimately, it may very well turn out that the majority, if not all, of the NLR integrated domains have lost their biochemical activities and have evolved into decoys. Also, it is possible that the sensor domain has already evolved into a decoy prior to recombination into a NLR. Nonetheless, further genetic and biochemical experiments are required to determine whether sensor domains of NLR-SDs are decoys or biochemically functional duplicates of their ancestral ETs.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Significant advances were recently achieved in the bidirectional control of upper limb neuroprostheses , howeverSince the long term goal is to implement a hardware version of the neuromorphic neurons, we modeled the mechanoreceptors as Izhikevich neurons, a model which at the same time is computationally lean and takes into account the effects of firing rate adaptation that are particularly relevant in mechanoreceptors. Parameters were tuned so to achieve a match with Slowly Adapting (SA) mechanoreceptors dynamics in primates . The outThe fingertip was then repeatedly presented with ten different daily-life textures such as textiles, polymeric tissues, glass or paper. FA spike trains were used to identify onset and offset of contact between fingertip and stimuli, whereas SA spike trains were used to decode the stimulus presented. We applied two decoding procedures: one based on the combination of firing rate and inter-spike interval Fano factor, and the other one based on Victor-Purpura distance . Error r


Introduction: Older adults are vulnerable to disproportionately higher morbidity following disasters. Reasons for this vulnerability are multifaceted and vary by disaster type as well as patient comorbidities. Efforts to mitigate this increased morbidity require identification of at-risk older adults who can be targeted for intervention.Methods: A PubMed search was performed using the search terms “geriatric, disaster” and “morbidity, disaster” to identify published articles that reported variables associated with increased morbidity of older adults during and after disasters. A review of article titles and abstracts was then conducted to identify those articles that contained evidence-based variables that render older adults vulnerable to poor health outcomes during disasters.Results: A total of 233 studies was initially identified. After applying exclusion criteria, nine studies were chosen for the comprehensive review. Based on the synthesis of the literature, factors were identified that were repeatedly associated with morbidity and mortality among older adults during and shortly after disasters.Conclusion: Older adults, especially those with multiple co-morbidities, are at risk of increased morbidity after disasters and catastrophic events. Factors such as the need for prescription medications, low social support, visual and hearing impairment, impaired mobility, and poor economic status are associated with an increased risk of morbidity. Older adults are especially vulnerable to disaster-related morbidity.Vulnerability of Community-Residing Older Adults to Disasters,,,,,,,Across the United States (US), the average age of the population continues to increase, with the majority of older adults living in the community with varying levels of social support. ,,For example, prior to Hurricanes Katrina and Rita in 2005, adults aged 60 or older made up only 15% of the population of New Orleans, Louisiana During a major disaster, a region’s medical resources may be overwhelmed. It is usually not possible for resources from out of state, such as disaster medical teams, to begin delivery of scene care within the first 72 hours of a disaster To identify factors associated with increased morbidity of older adults during and shortly after disasters, we reviewed published articles in this domain.The National Library of Medicine's MEDLINE database was searched utilizing the PubMed search engine. Specifically, the search terms “geriatric, disaster” and “morbidity, disaster” were used to identify published articles that reported variables associated with increased morbidity of older adults during and after disasters. A review of article titles and abstracts was then conducted between January to March 2014 to identify those articles that contained evidence-based variables that render older adults vulnerable to poor health outcomes during disasters.Articles that studied a population aged 55 and older were included to capture a larger proportion of the literature. Articles were excluded for the following reasons: 1) no data pertaining to adults >55; 2) no data pertaining to a disaster; 3) review articles without original data; 4) studies of nursing home patients; and 5) no risk factors identified that were present prior to the disaster. Please refer to Figure 1 for further detail.We initially identified 233 studies. After applying the exclusion criteria, nine studies were chosen for review. These included quantitative analyses, qualitative analyses, policy analyses, and expert observations from specific disasters. The references of each identified study were scanned to identify additional studies of interest. Based on the synthesis of the literature, we identified factors that were repeatedly associated with morbidity and mortality among older adults during and shortly after disasters.,,,,,,,Several studies specifically identified evidence-based vulnerability factors that render older adults vulnerable to poor health outcomes during disasters (Table 1). The most frequently cited risk factors for increased morbidity in community-residing older adults after a disaster were the presence of multiple co-morbiditiesThe top discharge diagnosis from one clinic in Louisiana after Hurricane Katrina was medication refills; other frequent visits were due to exacerbations of chronic diseases To minimize the impact of disasters on older adults, it is essential to first conduct an accurate community-based assessment of older adults’ vulnerabilities and utilize this assessment for improved planning Older adults living in the community are a heterogeneous group with varying levels of physical and mental health needs, social and family support, living arrangements, and incomes In addition to the general population of community-residing older adults in whom the prevalence of pre-existing vulnerabilities has not been determined, “frail” older adults, defined as those with serious, chronic health conditions such as heart disease, have not received significant consideration. Determining the prevalence of vulnerability amongst this population is especially important given that they are more likely than younger and healthier residents to need extra assistance with evacuations and recovery from disasters The response of public safety officials and first responders would also be enhanced by the study of the prevalence of vulnerability factors. In addition, if these vulnerabilities were known beforehand, the same public safety personnel could work to mitigate these factors. Public safety officials, such as the local fire department and emergency medical services (EMS), can serve as a vehicle for outreach and resource to deliver these needs Disaster-Relevant Research Conducted in Advance of a DisasterMuch research in disaster medicine is performed during the disaster and in real-time situations, with few resources for planning or an evidence-based approach Other experts in disaster medicine have also called for research to be conducted before disasters to inform evidence-based planning and response. Although disaster relief operations may be data driven, they are not necessarily evidence-based. This is especially the case when needs assessments are performed after disasters, yet no data exist regarding the prevalence of vulnerability factors. Options for strengthening evidence-based activities include rigorously adhering to evidence-based interventions, using evidence-based tools to identify new approaches to problems of concern, and improving standards for evidence of effectiveness in disaster science and services Although not identified in the literature search as a vulnerability factor, it has been noted that electricity loss may put older adults at increased risk during disasters. Older adults are more vulnerable to hypothermia and hyperthermia during temperature extremes, and may suffer during energy loss in a climate controlled home. A loss of electrical power also prevents the use of needed medical equipment, such as nebulizers and home oxygen therapy Utilization of Existing Databases for Disaster ResearchHowever, determining the prevalence of their vulnerabilities remains a challenge.As mentioned above, research conducted during disasters is methodologically challenging and often does not provide the data necessary for planning and response. Adequate planning and educational tools require information regarding the prevalence of risk factors. At a 2009 National Academies meeting, consensus was reached regarding the importance of mining existing data for insights and leveraging ongoing epidemiological and population surveys for pre-disaster baseline data Previous research has identified characteristics of community-residing older adults that predispose them to increased morbidity during disasters Through the utilization of pre-existing databases, it may be possible to identify vulnerabilities of older adults prior to disasters. Ideally, the databases would be large and detailed enough to identify by neighborhood or community, which localities have the greatest need for specific services.ConclusionOlder adults are at a known risk for increased morbidity during and shortly after disasters and catastrophic events, especially in the presence of multiple co-morbidities such as hypertension, diabetes, stroke, and heart disease. Other factors, such as the need for prescription medications, low social support, visual and hearing impairment, impaired mobility, and poor economic status, have also been associated with an increased risk of negative health outcomes. The utilization of existing databases of older adults’ vulnerabilities and the measurement of the prevalence of these risk factors in the community may enhance disaster research and response methodologies and decrease disaster-related morbidities.


Genetic analyses of autoimmune diseases have revealed hundreds of disease-associated DNA variants, but the identity and function of the causal variants are understudied and warrant deeper mechanistic studies. Here, we highlight methods for deciphering how alleles that are associated with autoimmune disease alter the human immune system, and suggest strategies for future autoimmune genetic research. The genetic mechanisms underlying human autoimmune diseases remain elusive for many reasons: the causal alleles or genes are often not known; the effects of each allele on gene function have often not been elucidated, especially as most alleles lie in less-well-understood non-coding regions; the impact of individual variants may be weak; and unknown environmental factors may be needed to provoke the effects of specific alleles. To address these deficiencies in our knowledge, studies of risk alleles will first need to define the roles of candidate variants in specific immune responses, and in time, in disease initiation, maintenance, and progression. Here, we highlight recent systematic approaches for discovering how human common genetic variants and disease-associated risk alleles alter the molecular and cellular functions of immune cells.Three recent studies have sought to reveal the contributions of both genetics and the environment on abundance and activation markers of different cell populations in the blood and their phenotypes. Orrù et al.  analyzedcis, as well as CD27 and CD161 expression on T cells, generating a novel and concrete hypothesis for how rs1801274 alters the immune system in humans. Brodin et al. [In what is the largest bioresource for immune cell phenotypes, Roederer et al.  list ~78n et al.  also fouIRF7 to have trans effects on interferon-inducible genes exclusively during influenza infection [IFNB1 that causes a delayed alteration in the response to lipopolysaccharide [IL2RA SNP that affects its own expression only in activated T cells [trans expression quantitative trait loci (eQTLs) that are relevant to autoimmune diseases, including a lupus-associated SNP in IKZF1 that affects the expression of several relevant genes, including C1Q and five interferon-inducible genes. By teaching us how common alleles alter immune networks, these studies are generating testable hypotheses to describe human autoimmune pathogenesis that will guide research in the field and suggest new therapeutic concepts.Numerous studies have considered how common alleles alter cellular states, as measured by RNA or protein expression. Most studies have monitored gene expression under baseline conditions, but a few recent studies have focused on cells that are activated in response to specific environmental stimuli, such as ligation of the T-cell antigen receptor, treatment with cytokines, or treatment with pathogen components. Examples of interesting findings include: the rs12805435 variant that causes  T cells . In a meIn addition to analyzing gene expression, some studies have considered how disease alleles localize within open chromatin. Maurano et al.  interseccis elements and cognate factors are likely to be impacted by variations in the DNA. These hypotheses bring us closer to a mechanistic model of how variants affect gene expression in specific cells and conditions, which can then be validated using modern perturbation tools such as CRISPR-mediated mutagenesis. By finding out which transcription factor (TF)–DNA interactions are affected by risk alleles, we will be able to predict which pathways (upstream of the TF) should be targeted for restoration of normal immune functions.Farh et al.  aimed toThe above studies focus on healthy individuals to avoid the complex effects of disease and treatment. And yet, some variants may only show their effects in the unique context of the patient with disease, as a result of deregulated signaling, positive feedback loops, and cellular crosstalk between pathogenic cell types. Furthermore, the open chromatin of patients might reveal permissive areas where disease variants could reside. An analysis of gene expression and chromatin states in sorted cell types from patients should be performed to reveal disease-specific effects and to link these phenotypes with genotypes. To ensure that patients are controlled for the treatment of their disease, studies could piggyback on a clinical trial, during which patient treatments would typically be more uniform than those given to the general patient population. In the future, personalized therapeutics and prophylactic measures could target gene products or pathways on the basis of information derived from a patient’s cellular epigenomic landscape, as defined by open chromatin and the patient’s risk variants that localize to this open chromatin. Understanding the genetic architecture in autoimmunity is important to derive such target genes, and pathways and bioresources such as those outlined by Roederer et al.  could ai


Where and when planarian stem cells become committed to the myogenic lineage during regeneration, how the new muscle cells are integrated into the pre-existing muscle net, and the identity of the molecular pathway controlling the myogenic gene program are key aspects of planarian muscle regeneration that need to be addressed. Expression of the conserved transcription factor myoD has been recently demonstrated in putative myogenic progenitors. Moreover, recent studies suggest that differentiated muscle cells may provide positional information to planarian stem cells during regeneration. Here, I review the limited available knowledge on planarian muscle regeneration.The body-wall musculature of adult planarians consists of intricately organized muscle fibers, which after amputation are regenerated rapidly and with great precision through the proliferation and differentiation of pluripotent stem cells. These traits make the planarian body-wall musculature a potentially useful model for the study of cell proliferation, differentiation, and pattern formation. Planarian body-wall muscle shows some ambiguous features common to both skeletal and smooth muscle cells. However, its skeletal nature is implied by the expression of skeletal myosin heavy-chain genes and the myogenic transcription factor  Pax7 and have been shown in several studies to be essential for skeletal muscle regeneration  they can regenerate an entire animal from a tiny portion of the body; and (ii) can do so thanks to the presence of a population of adult somatic pluripotent stem cells . The inner longitudinal fibers are thicker than the outer ones. These layers are compressed within a region of 7–12 μm thick below the epidermis  genes encoding 2 different muscle fiber types have been identified in freshwater planarians. One is expressed in the muscle fibers of the pharynx, the muscles surrounding the gastrodermis, in a few scattered cells throughout the body-wall, and in some muscle fibers in the mesenchyme at the base of the pharynx. The other mhc gene is expressed in the subepidermal body-wall musculature and in the dorsoventral fibers  or peristaltic movements during food intake (pharynx and enteric muscle).Myosin proteins are highly conserved in all eukaryotic cells, in which they provide the motor force necessary for different kinds of movements, including cytokinesis, phagocytosis, organelle movement, and muscle contraction  exhibits several ambiguities: like vertebrate smooth muscle cells planarian muscle cells are mononucleated, and can range from 150–200 μm in length and 5–10 μm in width MacRae, . At the  Sarnat, . At the  Sarnat, . In some Sarnat, . These Z Sarnat, . Moreove Sarnat, .mhc genes are expressed in different muscle types with different physiological functions, phylogenetic analyses indicate that they are more similar to mhc genes in striated muscle from other animals, including vertebrates, than to smooth muscle-type mhc genes  and in Smedwi-1-positive cells in the mesenchyme surrounding this organ. These FoxA/Smedwi-1 cells are lineage-specific progenitors; RNAi silencing of FoxA inhibits the differentiation of a new pharynx during regeneration  that correspond to the body-wall musculature  and by the expression of genes and proteins associated with cell division including histone H2B, PCNA, phosphohistone H3, and  Cebrià, . myoD be Cebrià, , suggestPrevious studies based on morphological criteria and electron microscopy suggested that the first myogenic cells within the blastema are detectable on days 2–3 of regeneration Sauzin, . More rein situ hybridization for the myoD homolog also support this model: the first differentiating cells expressing these markers are seen at very early stages of regeneration in the pre-existing tissues adjacent to the blastema  blastema cells are naïve undifferentiated cells that are committed and differentiate within the blastema; or (ii) blastema cells are a heterogeneous population of cells which enter the blastema already committed to specific cell lineages. Recent data from different models favor the latter scenario , pluripotent neoblasts must differentiate into all missing cell types. This process needs to be tightly regulated to ensure differentiation into the exact cells types required in each territory. As many cell types and organs are differentially distributed along the anteroposterior (AP) and dorsoventral (DV) axes, neoblasts need to receive precise information about the specific tissues that are missing in the different regenerative contexts . Little is known about how positional identities are maintained and re-established during planarian regeneration. Some findings suggest that such positional information resides in differentiated cells (Kato et al., It will be interesting to test this model in future studies, for example by characterizing differences in PCG expression in specific muscle cells from different regions in various regenerative contexts, or by analyzing the regenerative capabilities of muscle-deficient planarians, if possible.in vivo study of how these stem cells become committed and differentiate into the muscle lineage during regeneration. Preliminary results suggest that neoblasts become committed to the myogenic lineage before they enter the regenerative blastema, supporting the recently proposed existence of specialized neoblasts. However, definitive experimental proof is required, preferably obtained by studying the distribution and dynamics of myoD-positive cells during regeneration. Similarly, the process by which new muscle fibers are integrated into the pre-existing muscle and the role of these pre-existing fibers as a scaffold during the regenerative process needs to be better characterized. Planarians are also useful for the study of the behavior of stem cells and muscle progenitors as animal's age, a line of investigation that may help explain the loss of muscle stem cells observed in aging mammals (Sousa-Victor et al., Freshwater planarians have a complex body-wall musculature that functions mainly as a skeletal support and during locomotion. In addition, the digestive system consists of a highly muscular tubular organ, the pharynx, and a highly ramified gut surrounded by enteric musculature that mediates peristaltic movements during food intake and digestion. At the gene expression level the 2 muscle types express distinct forms of myosin heavy-chain genes. Planarians can regenerate and renew any type of cell and tissue through the differentiation of neoblasts, a population of adult pluripotent stem cells. These animals thus constitute an ideal model for the myoD sufficient for the differentiation of the myogenic lineage? What about other important transcription factors such as Mef2 genes and pax7 required for myogenesis in other models? Also, the fact that mature muscle cells could provide with positional information to planarian stem cells may be of great relevance to understand the regulation of those stem cells, as the existence of a niche for their activity has not been shown so far in these animals.Therefore, future studies should address some of the important lacuna that we have in the field mostly related to the regulation of the planarian stem cells and muscle progenitors. How conserved is the myogenic program in planarian stem cells compared to other systems? Is planarian in vivo how stem cells and muscle progenitors could be differently regulated in these two scenarios (Adler and Sánchez Alvarado, Finally, recent studies have shown that, in mammals, stem cells may have different behaviors under homeostatic or regenerative scenarios meaning that different signals from their changing environments trigger specific behaviors in each situation (Donati and Watt, In conclusion, planarian muscle may represent an attractive paradigm in which to study basic aspects of regeneration including stem cell biology, pattern formation, and positional information with further implications for the field of regenerative medicine.The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.