Hydrogels are promising biomaterials because of their important qualities such as biocompatibility, biodegradability, hydrophilicity and non-toxicity. These qualities make hydrogels suitable for application in medical and pharmaceutical field. Recently, a tremendous growth of hydrogel application is seen, especially as gel and patch form, in transdermal drug delivery. This review mainly focuses on the types of hydrogels based on cross-linking and; secondly to describe the possible synthesis methods to design hydrogels for different pharmaceutical applications. The synthesis and chemistry of these hydrogels are discussed using specific pharmaceutical examples. The structure and water content in a typical hydrogel have also been discussed. Out of these, semisolid transdermal drug formulations such as creams, ointments, and gels are most commonly used to provide systemic effect by delivering the drug across the skin. Gel, among all, is most preferred because of their excellent appearance, fast drug release, desired consistency, ease of manufacturing and quality assessment, and considerable stability.2 Most importantly, gels can be modified in order to make it suitable for delivering drug in sustained manner.The formulations applied onto the skin surface are broadly classified in to two groups such as topical and transdermal. Topical formulations deliver drug to local area of skin without systemic exposure. On the other hand, transdermal formulations applied to the skin surface for the purpose of delivering and maintaining effective concentration of drug in the systemic circulation.3An ideal gel should satisfy the following three salient features:They must have at least two components i.e. the gelling agent and a fluid component.Each component should be continuous throughout the system.They should exhibit mechanical properties of the solid state.4 According to USP, gels are defined as a semisolid, either suspension of small inorganic particles or large organic molecules interpenetrated with liquid.5 In gel system a liquid phase is constrained within a rigid three dimensional polymeric network thereby exhibiting visco-elastic nature. Gels can be classified into hydrogels and organogels based on the liquid medium entrapped within the polymeric network. Hydrogels are composed of aqueous phase, whereas organogels are composed of aqueous phase along with organic phase.Gels are transparent or translucent semisolid formulations containing a high ratio of solvent/ gelling agent. In other word, gel can be defined as a semi-solid preparation composed of low concentrations (<15%) of gelator molecules.7 Polymer networks in hydrogel may absorb water from 10–20% (an arbitrary lower limit) up to thousands of times their dry weight.9Hydrogels are hydrophilic, three dimensional cross-linked polymer systems capable of imbibing large amounts of water or biological fluids between their polymeric chains to form aqueous semi-solid/solid gel networks.14Followings are the main features of hydrogels for which they are widely used in pharmaceutical applications:Both compositionally (such as glycosaminoglycans) and mechanically, hydrogels are similar to the native extracellular matrix, thus can serve as a supporting material for cells during tissue regeneration and carrier in delivering a therapeutic agent.It exhibits soft material nature, which encourages uptake of water and thereby forming hydrated yet solid materials, just like cells in the body. The elastic property (due to the presence of meshes of the networks) of fully swollen or hydrated hydrogels is found to be minimizing irritation to the surrounding tissues after implantation. Their hydrophilic and cross-linked property imparts excellent biocompatibility. Low interfacial tension between the hydrogel surface and body fluid minimizes protein adsorption and cell adhesion, which reduces the chances of a negative immune reaction.Mucoadhesive and bioadhesive characteristics of many polymers used in hydrogel preparations (e.g. polyacrylic acid (PAA), polyethylene glycol (PEG), and polyvinyl alcohol (PVA)) enhance drug residence time on the skin/plasma membrane, leading to increase in tissue permeability.Presence of water in hydrogels plays an important role in the overall permeation of active ingredients into and out of the gel. Water can be associated to any hydrogel structure in following ways as shown in The types of water associated with hydrogel are:Bound water (primary and secondary)Semibound waterInterstitial water Free water or bulk water15When a dry hydrogel comes in contact with water, absorption of water into its matrix starts. At first water molecules entering the matrix will directly attach to the hydrophilic groups, forming hydrophilic bound water or primary bound water. The polymeric network swells because of complete hydration of the polar groups that resulted in the exposer of hydrophobic groups. These exposed groups also interact with water molecules, leading to hydrophobically-bound water, or ‘secondary bound water’. The combination of primary and secondary bound water is often called the bound water. This water is considered as integral part of hydrogel structure and can only be separated out from the hydrogel under extreme conditions. After the saturation of hydrophilic and hydrophobic groups, the additional water that is absorbed due to the osmotic driving force of the network chains is called free water or bulk water. Between the bound water, present on polymeric monomer surface, and free water, there is a presence of water layer called semibound water. Interstitial water is present in the interstices of hydrated polymeric network that is trapped physically but not attached to hydrogel network.Pharmaceutical hydrogels may be classified on the basis of their type of cross-linking or microstructure into following types :17 Because the network formation by all of these interactions is purely physical, gel formation can be reversed.In the preparation of physical gel, the selection of polymer is crucial and it depends on the two primary criteria; interaction among the chain must be strong enough to form semi-permanent junction in the molecular network and the network should hold large amount of water molecules inside it. The forces involved in the physical gel formation are hydrophobic, electrostatic, and hydrogen bonding between polymer chains.18The mechanism involved in the sol-gel and gel-sol conversion of thermoreversible gel is based on hydrophobic interaction. The polymers showing hydrophobic interaction must be having both hydrophilic and hydrophobic domain, called as amphiphiles. Such amphiphiles are water soluble at low temperature. However, following increased temperature the hydrophobic domains (gelators) aggregate to minimize the hydrophobic surface area contacting the bulk water. This reduces the amount of structured water surrounding the hydrophobic domains and maximizing the solvent entropy. The gelation temperature depends on various parameters such as the concentration of the polymer, the length of the hydrophobic block, and the chemical structure of the polymer.19 Gelation is also dependent on the concentration of PX molecules in solution. At very low concentrations, PX molecules exist as monomers in solution. Increasing PX concentration up to 10−4% to 10−5% (w/w) leads to a critical micelle concentration (CMC), where spherical micelles started to built up. Further increase in PX concentration leads to a tightly packed system with a gel consistency.21 Between 20-30% (w/v) aqueous solution of PX 407 at cold temperature (4-5°C) forms clear liquid and get transformed to gel at body temperature (37°C).Poloxamer (PX) is such a polymer which shows thermoreversible property in aqueous solutions. PX consists of hydrophilic poly (ethylene oxide) (PEO) and hydrophobic poly(propylene oxide) (PPO) blocks arranged in a tri-block structure as PEO–PPO–PEO [19]. The gelation of PX depends upon both temperature and concentration and the total gelling process is typically divided into two steps . The fir22 Erukova et al. (2000) observed that PX facilitated the permeation of comparatively large molecules, including 2-n-undecylmalonic acid and doxorubicin, across lipid bilayers, whereas the permeation of small solutes (such as ammonium and acetic acid) remained unaffected. They also observed that PX accelerated the translocation of large hydrophobic anions (tetraphenylborate). They attributed the above effect of PX with the content of PEO units i.e. it is enhanced when the portion of PEO block in the copolymer is increased.23 Gels and niosomal gels of meloxicam were developed using polymers such as PX 407, Chitosan and Carbopol-934. The PX 407 gel or niosomal PX 407 gel showed the superior drug release over the other formulations. Between the two types of gel of PX 407, niosomal gels exhibited superiority over conventional gels for anti-inflammatory test.24 Thermosensitive gels of drugs such as indomethacin,25 adriamycin and 5-flurouracil,26 mitomycin C,27 and ketoprofen28 were successfully developed and studied. The gel formed from PX alone (as in case of above) leads to relatively rapid diffusion of drugs out of the hydrophilic gel matrix by the dilution of water that entered the gel matrix.29 To circumvent above drawback many researchers have used combination of polymers to reduce the fast entry of water and subsequently diffusion of drug out of gel structure. In addition, it will retain the gel structure for longer period of time and thereby sustain the drug release.Release of the model drug benzoic acid was found to be decreased with the increase in PX 407 concentration. This is probably due to an increase in size and number of micelles leading to decrease in size and number of aqueous channels. Further the modification of benzoic acid to more lipophilic moiety p-hydroxybenzoic acid causes a decrease in its release rate. This could be due to the greater partitioning of lipophilic derivatives into the micellar region within the gel structure.30 HPMC K100M (2% w/v) and PX 407 (20% w/v) based procaine bioadhesive gels were prepared and the drug release was found to be increased with the increase in drug concentration from 1.5% to 3.5% (w/w) in the gels and the temperature of surrounding solutions from 28°C to 42°C.31 Pranoprofen release from HPMC and PX 407 based bioadhesive gel was increased with the increase in temperature, which was attributed to increased in activation energy of the drug.33 Similarly, Shin et al. (2004) successfully prepared lidocaine HCl gel using HPMC (2% w/v) and PX 407 (20% w/v) for analgesic activity.34 We have successfully prepared and characterized bioadhesive hydrogels of aceclofenac and metoprolol succinate containing various concentrations of HPMC (0.5-4% w/w) and PX 407 (15-30% w/w).36Based on the above fact thermoreversible gels of meloxicam were prepared using PX 407 and PX 188 (20-30% w/w) in combination with different additives such polyvinylmethylether maleic anhydride copolymer, HPMC, PE-400, DMSO, sodium chloride. Among investigated gel bases, PX 407-HPMC gel was found to be ideal due to its gel strength (1.560±0.0135 N), viscosity (312.3±2.06 cP) and release characteristics.37 or PLGA-PEG-PLGA38 etc. Compared to PX systems, PL(G)A-based triblock gelators exhibit better biodegradability, higher gelation temperatures , and sustained drug release.39 Another example of symmetric triblock polymer is Poly (N-isopropylacrylamide) (PNIPAM)-poly (phosphorylcholine)-PNIPAM, which form gels at 6-7 wt % when the temperature exceeded 32°C (is the phase transition temperature of PNIPAM).40 PNIPAM grafted with other polymers such as hyaluronic acid and chitosan were developed successfully for the sustained release of riboflavin41 and 5-fluorouracil,42 respectively. Asymmetric triblock copolymer formed of PEG, PLA, and poly (L-glutamic acid) were also developed and observed that the L-glutamic acid block permits selective modification with specific targeting groups such as the cell-adhesive RGD peptide.43A range of other synthetic amphiphilic thermally gelling polymers have been investigated. Based on the position of hydrophilic and hydrophobic blocks they may be classified in to two categories; symmetric and asymmetric. Symmetric means the presence of hydrophobic block at the center and hydrophilic blocks on either side and vice versa. Examples of this category are PX (discussed in the previous section), PEG- poly (lactide-co-glycolide) (PLGA)-PEG44 Chitosan grafted with PEG (approximately 40 wt %) based thermoreversible gel was prepared and studied for bovine serum albumin (BSA) release. This gel showed an initial burst release for 5 h and afterwards a steady linear release of BSA for a period of approximately 70 h.45 Liang et al. (2004) successfully used thermosensitive methylcellulose to thermally gel aqueous alginate solution blended with various salts such as CaCl2, Na2HPO4, and NaCl for the delivery of BSA.46 Similar thermally triggered transitions have also been exhibited by hydroxypropylcellulose.47Reverse thermal gelation is also evident from some natural polymers. A biocompatible, parenteral gel composed of chitosan solutions and glycerol-2-phosphate was prepared and the gel was found to be exhibited sol-gel transition at a temperature close to 37°C.48 A mixture of quaternized chitosan (N-[(2-hydroxy-3-trimethylammonium) propyl] chitosan chloride (HTCC)) and glycerophosphate (GP) forms an ionically cross-linked gel at 37°C which can release doxorubicin hydrochloride as a function of pH.49 At acidic condition and physiological condition these hydrogel dissolved and released drug quickly, while it released drug slowly at neutral or basic conditions.Charge interactions which lead to the formation of hydrogel may occur in two ways; (i) between a polymer and an oppositely charged small molecule as linker, and (ii) between two polymers of opposite charge . This ty50 Another approach, so called ionic-complementarity, uses peptide of alternating positive and negative charge distribution leading to peptide self-assembly. These peptides assume β-sheet secondary structure predominantly, and can form hydrogels. The advantage of this approach is that nanoscopic and/or macroscopic structures with great stability and functionality can be developed by varying peptide concentration, pH, presence of salts, and time.51The charged interactions have been widely investigated for cross-linking in situ gelling hydrogels. The main advantage of this interaction is that biodegradation can occur as ionic species in extracellular fluid which bind competitively with the gel components and as a result decross-linking of network happen. In addition, this approach can also be used to cross-link microparticle or nanoparticle gels to equip it with suitable drug delivery properties. For instance anionic (methacrylic acid (MAA)) and cationic (dimethylaminoethyl methacrylate (DMAEMA)) polymers coated hydroxyethyl methacrylate-derivatized dextran microspheres exhibit spontaneous gelation upon mixing due to ionic complex formation between the oppositely charged microparticles.16 Its distinction from electrostatic interactions is based on the MW of molecules. The PEC is formed between larger molecules with a broad MW range and the association is stronger than other secondary binding interactions like hydrogen bonding (H-bonding) or van der Waals interactions. For example formation of PEC between chitosan, a polycation, with polyanions such as proteins , polysaccharides  and synthetic polymers (e.g. PAA) were reported.52PECs are formed by the electrostatic interactions between two oppositely charged polyelectrolytes in an aqueous solution as shown as in 53 Another chitosan based PEC hydrogel with improved viscoe-lastic behavior was developed using low concentration (0.1 to 0.8%) of chitosan and constant gelatin concentration of 1%.54 Chang et al. (2014) successfully developed PEC of chitosan and poly-(γ-glutamic acid) (γ-PGA) to promote new bone formation in the alveolar socket following tooth extraction.55 With same composition, a cytocompatible PEC was developed for biological application.56 A PEC composed of alginate and chitosan was developed and found to be having improved thermal, chemical, and mechanical properties and stability.57 Sometimes, same polymers having opposite charges, fraction is modified to opposite charge by chemical processes, can also be used to prepare PEC hydrogel. One of such hydrogel was developed with phosphorylated chitosan (a polycation) and chitosan (a polyanion) as an osteoblast carrier. These hydrogels exhibited three-dimensional hierarchically-porous structure and good cyto-biocompatibility for osteoblasts in vitro.58 Based on above approach, another novel PEC encapsulated dexamethasone hydrogel was prepared using polycationic N-trimethyl chitosan and polyanionic N-carboxymethyl chitosan to target distal intestine.59 Chitosan, in many drug delivery systems, present as unionized amine form at the neutral/alkaline pH of physiological application sites, while the anionic polyelectrolyte is in the ionized form. In this form to stabilize any PEC is a challenging task, but it can be overcome by using modified chitosan such as quaternized chitosan. This modified chitosan along with PAA were used to prepare PEC hydrogels with improved mechanical properties by in situ polymerization of acrylic acid monomers in the concentrated quaternized chitosan solution.60Chitosan based PEC of metoprolol tartrate were developed in the form of beads and hydrogels by interaction of cationic chitosan with anionic sodium alginate, carboxymethyl cellulose sodium and k-carrageenan using simple ionic gelation technique. Among all the combinations, PEC of chitosan and carboxymethylcellulose sodium exhibited least in vitro drug release and was able to extend the release of metoprolol tartrate up to 12 h.61 which is quite common and hinders the formation of hydrogels. Precipitation can be prevented weakening the electrostatic attraction by the addition of salts, such as NaCl.16The advantage of PEC is that it is formed without the use of organic precursors, catalysts, or reactive agents, avoiding the concern about safety in the body. During complexation, polyelectrolytes can either coacervate, or form a more or less compact hydrogel. However, if ionic interactions are too strong, precipitation can occur,62 or in combination with other polymers such as chitosan.52 In the earlier case crystallization plays a big part in the creation of cross-links between PVA chains during lyophilization. The cross-linking is possible due to the existence of regular pendant hydroxyl groups on PVA that are able to form crystallites by strong inter-chain H-bonding. The crystals formed by this method are generally considered to have a two-molecule monoclinic unit cell, with hydroxyl groups randomly placed on either side of both polymer chains as shown in 63Hydrogels by H-bonding interactions can be formed by freeze-thaw cycles using PVA aloneet al. (2009) reported that with increasing number of thermal cycles resulting in the formation of the strongest cryogels because the number of intermolecular hydrogen bonding increases and molecular chains aggregate.64 They also reported that the increase in thawing rate from 2 (0.333°C/min) to 5 (0.067°C/min) exhibited the same effect on the gel’s elastic properties. Meenakshi, and Ahuja, (2015) found that 4 freeze-thaw cycles are optimum for the preparation of composite cryogels of metronidazole with carboxymethyl tamarind kernel polysaccharide and PVA. This cryogel provided 75.77% of drug release over a period of 6h and had better thermal stability.65Processing parameters such as the concentration, the number of thermal cycles, and the thawing rate affect the mechanical behavior of cryogel. Pazos 16 These interactions act as cross-linking sites of the hydrogel. Addition of another polymer to PVA negatively affects the formation of PVA crystallites thereby forming hydrogels of less ordered structures. Chitosan-PVA polymer blend is one such example of polymer mixture, where increasing chitosan concentration in the blend reduces the crystallinity of hydrogel.52 The addition of chitosan decreased maximum strength and thermal stability of PVA cryogel containing minocycline and gentamycin, while it increased the swelling ability, elasticity and porosity of cryogel.67 Contrary to above, Paduraru et al. (2012) reported that strength of the cryogel increased by increasing the microcrystalline cellulose content from 0 to 50 % wt.68In case of composite cryogel, mixture of polymers forms junction points in the form of crystallites and inter-polymer interaction after a series of freeze–thaw cycles.69 The main advantage of above mixture of polymers is the excellent biocompatibility offered by them due to the absence of chemical cross-linkers. However, the problem associated with it is the breaking of H-bonded networks over a few hours in vivo due to influx of water, which restricting their use to short-acting drug release systems.18H-bonding can also be formed with blend of two or more natural polymers such as hyaluronic acid-methylcellulose, gelatin-agar, and starch-carboxymethyl cellulose. The formation of H-bonding interactions between the polymer chains along with the compatible geometry resulted in the enhanced visco-elastic properties of the hydrogel.70 It may be homo-stereocomplexation or hetero-stereocomplexation. In former case the complex formation occurs between same molecules but with different stereochemistry. For example, interaction between polylactide blocks with L- and D-stereochemistry.70 Another well known example of stereocomplexation is the one between isotactic and syndiotactic poly(methyl methacrylate) (PMMA).71 Hetero-stereocomplexation occurs between two different molecules with different stereochemistry such as D-PLA and L-configured peptides leuproide, a luteinizing hormone-releasing hormone.72 Natural polymers such as dextran precursors can be grafted to L-lactide and D-lactide oligomers leading to the spontaneous gelation in water. It provided an excellent biocompatibility and biodegradability.73Stereocomplexation is a synergistic interaction between polymer chains or small molecules of the same chemical composition, occasionally with different chemical composition, but different stereochemistry and stereocomplex formation occur due to the interaction between polymers having different tacticities or configurations.Like H-bond interactions, stereocomplexation do not need harsh organic solvents and chemical cross-linkers. Main limitation of stereocomplexation is the restricted range of polymer compositions available which can be used i.e. even small changes in the stereochemistry or else changing the composition of matter can significantly weaken or altogether eliminate the stereochemical interaction.74Supramolecular chemistry is based on the association of two or more chemical species held together by intermolecular forces. The intermolecular forces includes electrostatic interactions, H-bonding, metal coordination, π- π interaction, Van der Waals forces etc. and the important methods by which supermolecules formed are; molecular self-assembly, folding, host-guest interaction (e.g. inclusion complex), recognization and complexation and mechanical-interlocking.75 Similarly, β-cyclodextrin can be used to gel PPO-grafted dextran into a hydrogel.76 Self-assembled systems have also been reported that apply both hydrophobic interactions and supermolecular chemistry to facilitate the formation of denser and/or more stable hydrogel networks. A self-assembled system, PEO-poly(R-3-hydroxybutyrate) (PHB)-PEO triblock copolymers, was complexed with α-cyclodextrin to form strong hydrogel networks. The cross-linking was accomplished by (a) the thermal gelation of the hydrophobic PHB segments and (b) the inclusion complexes formed between the PEO segments and cyclodextrin. This system is capable of releasing fluorescein isothiocyanate-dextran for up to one month.77This approach has wide application such as material technology, catalysis, data storage and processing and in the preparation of medicines. The most common type of cross-linking interaction used to prepare hydrogels is the formation of inclusion complexes. For example, formation of a reversible hydrogel complex between PEO polymers and α-cyclodextrins.52 In contrast, chemically cross-linked hydrogels allows absorption of water and/or bioactive compounds without dissolution and permits drug release by diffusion.18Physically cross-linked hydrogels are prepared without the use of cross-linking entities or chemical modification. Despite of the above advantage, it is inflexible towards variables such as gelation time, gel pore size, chemical functionalization, and degradation or dissolution, leading to inconsistent performance in vivo.Chemical cross-linking method uses covalent bonding between polymer chains in order to produce permanent hydrogel. The cross-link formation was carried out by the addition of small cross-linkers molecules, polymer-polymer conjugation, photosensitive agents or by enzyme catalyzed reaction.78 Small cross-linkers can be of two types; bi-functional molecules or drug molecules. In the former case drug molecule is entrapped within the hydrogel formed due to the interaction between bi-functional molecule and polymer. Moreover, drug molecule having two functional group covalently bonded with polymeric chains to form hydrogel, which avoids the use of cross-linkers. This approach is limited to drug in which two reactive functional groups are available. One such drug molecule is primaquine (a di-amino drug), which has been used to cross-link periodate-oxidized gum arabic into a hydrogel through the rapid formation of Schiff bases between the amine groups and the aldehyde groups present in the drug and polymer, respectively.79Preparation of small-molecule cross linking hydrogels at the minimum requires one polymer and a small molecule as cross-linker in an appropriate solvent. Cross-linkers are molecules with at least two reactive functional groups that allow the formation of bridges between polymeric chains.46 forms covalent imine bonds with the amino groups of chitosan via Schiff reaction.80 In other cases, dextran-tyramine81 and hyaluronic acid-tyramine82 covalently bonded by using horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) as cross-linkers to form hydrogels with improved controllable gelation times ranging from 5 s to 9 min according to the reactant concentrations used. A general disadvantage of each of these small-molecule cross-linking methods is the potential toxicity of residual unreacted cross-linker agents in vivo. For instance, glutaraldehyde and glyoxal are known to be neurotoxic and mutagenic, respectively.80The simplest form of the cross-linking takes place between aldehyde and amino groups to form Schiff base. For example, dialdehyde such as glyoxal and particularly glutaraldehyde83 It was also reported that genipin bind polymers, such as chitosan and gelatin with biological tissues covalently.84 Furthermore, amino-terminated groups containing molecules such as PEG, N,O-carboxymethyl chitosan,85 and BSA86 have been cross-linked to genipin to form hydrogels with flexible dissolution rates from 3 min to more than 100 days. Even though genipin shows good biocompatibility, it is still liable to negatively interact with encapsulated drugs, an unavoidable problem for gelation in the presence of a therapeutic.Genipin  widely used as a cross-linking agent alternative to dialdehydes due to its biocompatibility.87 propanolol HCl,88 5-fluorouracil,89 indomethacin from gel beads for the control release.90This approach used to develop transdermal drug delivery systems for the delivery of oxprenolol HCl,In this type of hydrogel, the cross-linking reaction occurs between a structural unit of a polymeric chain and a structural unit of another polymeric chain of another type. Therefore, polymers must be pre-functionalized with reactive functional groups. Several types of covalent linkages can be made depending on the desired speed of cross-linking, selection of targeted reactive functional groups, and biodegradability of the resulting conjugates.91 Another example is the formation of a hydrazone bond between an aldehyde and a hydrazide which facilitates rapid cross-linking of gel precursors.92 Control release of tissue plasminogen activator and budesonide93 to the peritoneum was achieved by using hyaluronic acid cross-linking by hydrazone bonds. Similar approaches also been used to design poly  for the effective delivery of osteoblasts and growth factors.94Widely investigated in situ cross-linking phenomenon is Michael addition between a nucleophile (i.e. an amine or a thiol) and a vinyl group. For example, cross-linking of vinyl sulfone-functionalized dextrans with thiolated PEG. This type of approach provides the flexibility in forming multiple types of bonds, relative biological inertness and finally rapid formation of hydrogel.The major advantages of this approach are the elimination of cross-linker molecules and in situ formation of covalently bonded hydrogels. The main limitation of this approach is the requirement for significant polymeric modification to link functional group to polymeric chain. In addition, the pre-gel polymers are often themselves somewhat cytotoxic, even when prepared from highly biocompatible polymer precursors.3) to polymeric chain of chitosan. After its exposure to UV light, the azide group is converted to nitrene group (R-N:) which binds free amino groups of chitosan leading to the in situ formation of hydrogel within a minute.95 Photo cross-linked hydrogel can also be formed between the polymers. One such example is thermo-sensitive chitosan-pluronic hydrogel, where both the polymers were functionalized with photo sensitive acrylate groups (CH2=CHCOO−) by UV irradiation. The resulted cross-linked polymer formed a physical network at temperatures above the lower critical solution temperature (LCST) and the same exhibited the ability to release encapsulated human growth hormone (hGH) in sustained manner.96 The above combination was also used to deliver plasmid DNA.97 Another chitosan-PEG-based hydrogel was developed by modifying chitosan with photoreactive azidobenzoic acid and PEG with arginylglycylaspartic acid peptide. Upon UV illumination, a free-radical photo-initiated polymerization took place that led to the formation of hydrogel in situ. Resulted hydrogel helped in targeting the injured myocardium for the delivery of growth factors and cells.98Formation of hydrogels based on photo cross-linking depends on the presence of photo sensitive functional groups. By linking a photo sensitive functional group to a polymer enables it to form cross-linkages upon irradiation with light such as UV light. Chitosan is one such polymer which was studied more compared to other polymers. A photo cross-linked chitosan hydrogel was developed by incorporating azide groups ,106 Yung et al. (2007) have successfully developed thermally stable and biocompatible gelatin hydrogels cross-linked by microbial TG (mTG), which is capable of delivering encapsulated regenerative cells (HEK293) in a controlled manner.100 The same group tested the diffusion of anticancer drug, interleukin-2, from mTG cross-linked gelatin and found that the resulted hydrogels are not only cyto-compatible but also have potential to be used as sustained drug release devices.101 Between animal derived tissue TG (tTG) and recombinant human TG (hTG) enzymes used for cross-linking of two classes of protein polymers containing either lysine or glutamine under physiological conditions, tTG completed the cross-linking faster (within 2 min) compared to hTG.102TG belong to thiol group of enzymes which catalyze the formation of highly resistant covalent bonds between a free amine group of a protein or peptide-bound lysine and the g-carboxamide group of a protein or peptide-bound glutamine.2O2. In this reaction the HRP promptly combines with H2O2 and the resulted complex can oxidize hydroxyphenyl groups present in compound such as tyramine and tyrosine.106 Recently, Kim et al. (2011) developed HRP catalyzed injectable tyramine modified hyaluronic acid (HA-Tyr) hydrogels in two steps. In the first step, HA-Tyr conjugate was synthesized by amide bond formation between carboxyl groups of HA and amine groups of tyramine and in the subsequent step HA-Tyr hydrogels were prepared by radical cross-linking reaction using HRP and H2O2. This hydrogel was used to deliver dexamethasone intra-articularly for the treatment of rheumatoid arthritis.103 An enzymatically cross-linked injectable hydrogel was developed from chitosan derivatives, chitosan-glycolic acid, and phloretic acid using HRP and H2O2.104 This hydrogel can be formed (gelation time) from 10 sec to 4 min by using the polymer concentration from 3 to 1% and has potential for cartilage tissue engineering.HRP and soy bean peroxidase are the most commonly used peroxidase enzymes in the formation of hydrogel. They catalyze the conjugation of phenol and aniline derivatives in the presence of substrate Het al. (2002) developed gelatin gel by two methods namely; (i) cooling the gelatin solution and (ii) tyrosinase-catalyzed hydrogel formation and observed that the second method demonstrated better mechanical properties which can be exploited for medical and industrial applications.107 Gelatin-chitosan gels were developed using both TG and tyrosinase for comparison and following observations were made; (i) chitosan was not required for transglutaminase-catalyzed gel formation, although gel formation was faster and the resulted gel was stronger in its presence, (ii) TG-catalyzed gelatin-chitosan gels lost the ability to undergo thermally reversible transitions, (iii) tyrosinase-catalyzed gelatin-chitosan gels were considerably weaker than transglutaminase-catalyzed gels, but can be strengthened by cooling below gel-point of gelatin.105Tyrosinases are oxidative enzymes that convert accessible tyrosine residues of proteins (e.g. gelatin) into reactive o-quinone moiety, which can undergo non-enzymatic reactions with available nucleophiles such amino groups of chitosan. Chen 109 IPNs are considered as “alloys” of cross-linked polymers, which are formed without any covalent bonds between them. These networks cannot be separated unless chemical bonds are broken.110An IPN may be defined as any material that contains two or more polymers in the network form in which one polymer is cross-linked in the presence of other.111 An IPN is different from other polymer combination because it has no viscoelastic property, and it swells, but does not dissolve in any solvent.112The polymers to be used in the preparation of IPN hydrogel must fulfill following three conditions; at least one polymer must be synthesized and/or cross-linked within the immediate presence of the other, both polymers should have similar kinetics, and there should not be dramatic phase separation between/among the polymers.114Based on the chemistry of preparation, IPN hydrogels can be classified into following types:Simultaneous IPN: when both the networks are synthesized simultaneously from the precursors at the same time by independent, non-interfering routs. Sequential IPN: it is formed by swelling of a single-network hydrogel into a solution containing the mixture of monomer, initiator and activator, with or without a cross-linker. Sequential IPNs are of two types namely, fully-IPN and semi-IPN based on the presence and absence of cross-linker, respectively.114According to the structure, IPN hydrogels can be classified into three categories:IPNs: formed by two juxtaposed networks, with many entanglements and physical interactions between them. Homo-IPNs: these are developed from the two polymers which are having independent networks, but have same structure. Semi- or Pseudo-IPNs: in this type one polymer has a linear instead of a network structure.2, SO3H, amines etc).8There are mainly two classes of polymers which are used to synthesize IPN hydrogels, including natural polymers and their derivatives such as polysaccharides and proteins, and synthetic polymers containing hydrophilic functional groups  developed IPN hydrogel membranes of prazosin hydrochloride using sodium alginate (SA) and PVA as polymers for transdermal delivery. The stiffness of the film was increased by the addition of cross-linker glutaraldehyde and both the stiffness and in vitro drug release property film depend on the concentrations of glutaraldehyde in membranes. The prepared IPN membranes extended prazosin hydrochloride release up to 24h, whereas membranes made up off SA and PVA alone showed fast drug release.117 Microspheres of theophylline were successfully prepared based on IPNs method using chitosan and methylcellulose as polymers and glutaraldehyde as a cross-linker. The encapsulation efficiency was found to be up to 82% and the extended drug release up to 12 h.118 Likewise, duration of 5-fluorouracil release was significantly sustained for up to 52 h from new thermo-sensitive gels in which a chitosan network is crosslinked with various concentrations of glutaraldehyde that interpenetrates PX gels.119IPN hydrogels of clarithromycin were synthesized by using three polymers such as chitosan, PAA and poly (vinyl pyrrolidone) (PVP) and two cross-linking agents . The resulted IPN hydrogels have good mucoadhesion property, which helped the gel to retain in gastric environment of stomach for prolonged period of time. As a result of that IPN hydrogel maintain antibiotic concentration in stomach for prolonged period of time, thereby used as a drug delivery system for treatment of 120 Furthermore, the cross-linking density, hydrogel porosity, and gel stiffness can be adjusted in IPN-based hydrogels according to the target application. The drawbacks are: (i) it has a problem with encapsulating a wide variety of therapeutic agents, especially sensitive bimolecular, and IPN, particularly sequential type, (ii) preparation requires the use of toxic agents such as initiator, activator and cross-linker in order to initiate or catalyze the polymerization or to catalyze the cross-linking.121 Difference between physical and chemical hydrogel and types of cross-linked hydrogel along with their polymer system and drug incorporated is presented in Multicomponent networks as IPNs have better mechanical strength and swelling/deswelling response compared to single-network hydrogels.6 Despite the wide use, hydrogels suffer significant disadvantages such as low mechanical strength and toughness, difficulties in handling, sterilization, syringability issues. Special structural configurations like slip-link networks, nanocomposite hydrogels, double network hydrogels, multi-functional cross-linked hydrogels, and homogeneous hydrogels can be synthesized to enhance mechanical strength and toughness of resulting hydrogels.122 These techniques should be followed by a suitable method of polymerization or cross-linking in order to make them desirable for suitable application. Another way to enhance mechanical property along with improved biocompatibility and physical property is to combine both physical and chemical cross-linking in a single hydrogel system.Over the past 50 years, there has been continuous progress in designing of hydrogels that led to numerous applications such as pharmaceutical, biomedical, agrochemicals, food industry, etc. In pharmaceutical therapeutic delivery, hydrogels are available in various dosage forms such as tablets, capsules, microspheres, transdermal films, wound dressings, etc. These hydrogels are also used to develop catheter, vascular grafts, semiocclusive dressings, mammary implants, transdermal drug delivery systems, scaffold for tissue engineering and medicated patches etc.124The syringability issue can be solved to a large extent by designing physical gelators which gel at lower polymer concentrations and at more precise gelation temperatures, like in-situ or thermosensitive gels, would reduce the risk of premature gelation inside the needle upon injection. Similarly, for covalently cross-linked hydrogels, the development of strategies to release cross-linker in a triggered manner inside the body, thereby minimizes the risk of syringe clogging, improve the localization of cross-linker release to minimize in vivo toxicity. This strategy is used to transform hydrogels in to “smart” materials that can respond to changes in their environment. In addition the hydrogel properties can be further modified by incorporation of micro or nano fillers. More recently, hydrogels have been modified into hydrogel microparticles for solubility enhancement purposes.In the present scenario, the focus needs to be shifted towards the development of innovative methods to prepare: (i) hydrophilic polymers of desirable functional groups, (ii) multifunctional/multiarm structures such as grafted or branched co-polymers and star polymers that would offer better properties and suit wider applications in the future.The author thanks management of GITAM University to provide necessary facilities and support.Not applicable.The author declares no conflicts of interest.


The goal of this study was to determine the extent of rod-, cone-, and melanopsin-mediated pupillary light reflex (PLR) abnormalities in diabetic patients who have non-proliferative diabetic retinopathy (NPDR). Fifty diabetic subjects who have different stages of NPDR and 25 age-equivalent, non-diabetic controls participated. PLRs were measured in response to full-field, brief-flash stimuli under conditions that target the rod, cone, and intrinsically-photosensitive (melanopsin) retinal ganglion cell pathways. Pupil responses were compared among the subjects groups using age-corrected linear mixed models. Compared to control, the mean baseline pupil diameters were significantly smaller for all patient groups in the dark  and for the moderate-severe NPDR group in the light (p = 0.003). Pairwise comparisons indicated: (1) the mean melanopsin-mediated PLR was significantly reduced in the mild and moderate-severe groups (both p < 0.001); (2) the mean cone-mediated PLR was reduced significantly in the moderate-severe group (p = 0.008); (3) no significant differences in the mean rod-mediated responses. The data indicate abnormalities in NPDR patients under conditions that separately assess pupil function driven by different photoreceptor classes. The results provide evidence for compromised neural function in these patients and provide a promising approach for quantifying their neural abnormalities. For all subjects, a comprehensive history was obtained from the medical record and a physical examination of each eye was performed by a retina specialist  with particular attention to the optic nerve, retina, and its vasculature. Diabetic subjects had best corrected visual acuity of 0.4 log MAR or better  and controls had visual acuity of 0.1 log MAR or better . Subjects were excluded if there was a history of, or if they presented with, ischemic optic neuropathy, other optic nerve disease, retinal artery occlusion, retinal vein occlusion, or glaucoma. Additionally, subjects with iris neovascularization, iris atrophy, or an asymmetrically shaped pupil were excluded. No subject had a history of neurologic events (cerebrovascular stroke or transient ischemic attack). The lens of each subject was graded by slit lamp examination using a subjective clinical scale that ranged from clear to 4+. Subjects with nuclear sclerotic, posterior subcapsular, or cortical lens opacities greater than 2+ were excluded. Although subjects with lens opacities of 2+ or less were included, we note that the effects of light scatter due to lens opacities are negligible because the stimuli were uniform full-field flashes. All 25 control subjects and 41 of the 50 patients were phakic; the remaining 9 patients were pseudophakic. No subject had cataract surgery in the 9 months prior to testing.For the diabetic subjects, the stage of NPDR was graded and the subjects were classified as diabetic with no NPDR (N = 17), mild NPDR (N = 16), moderate NPDR (N = 13), and severe NPDR (N = 4), based on the International Clinical Disease Severity Scale for DR2) flash was presented in the dark; (2) under the melanopsin paradigm, a short-wavelength , high luminance (450 cd/m2) flash was presented in the dark; (3) under the cone paradigm, a long-wavelength , moderate luminance (10 cd/m2) flash was presented against a rod-suppressing short-wavelength adapting field (6 cd/m2). All flashes were 1 sec in duration. Responses to a minimum of two flashes (separated by at least 15 sec for the rod and cone conditions and 60 sec for the melanopsin condition) were obtained and averaged for analysis. Prior to the stimulus presentation under the rod and melanopsin paradigms, the subjects were dark-adapted for 10 minutes, whereas the subjects were light adapted for 2 minutes to the rod-suppressing adapting field prior to stimulus presentation under the cone paradigm. Stimulus wavelength and luminance were verified with a spectroradiometer .An LED-driven ganzfeld system was used for stimulus generation and display . Details and images of the system are available elsewhere2815Data were analyzed using custom scripts programmed in MATLAB , which allowed for semi-automated analysis as described elsewhere15Pupil size was compared among the subject groups statistically using linear mixed models to account for covariates35362) presented in the dark is shown in the upper right panel for comparison to the short-wavelength flash used under the melanopsin condition. The response elicited by the long wavelength flash consisted of only the initial transient constriction, which was somewhat reduced in the mild and moderate-severe NPDR groups. For the long wavelength flash, there was no sustained response for any subject group and the traces for the control and patient groups are nearly superimposed in the 5 to 7 sec window following stimulus offset . Since a substantial sustained PLR was not obtained with the long wavelength flash, data from this condition will not be considered further.For the rod . Age adjusted, Tukey pairwise comparisons indicated significantly smaller light-adapted baseline pupil sizes only for the patients with moderate-severe NPDR compared to the control group . The mean (±SEM) dark- and light-adapted age-adjusted baseline pupil sizes (mm) for each group are given in As an additional consideration, several patients in the mild and moderate-severe NPDR groups had a history of treatment with focal laser therapy and/or anti-VEGF agents see . To evalThe patients’ rod-mediated , middle To evaluate the effects of treatment on the stimulus-driven PLR, sub-analyses were performed after excluding the 8 patients who had focal laser therapy ; row 9. The relationship between each PLR measure and patient characteristics including age, disease duration, and HbA1c percentage were examined by computing Pearson correlation coefficients. The relationships among these parameters were generally weak  and were not statistically significant after correcting for multiple correlations. There were also no significant differences between males and females within all subject groups for all PLR measurements .th percentile of the control range and “baseline” refers to the dark-adapted baseline for the melanopsin and rod conditions and the light-adapted baseline for the cone condition. Overall, 71% (12/17) of no the NPDR group, 75% (12/16) of the mild NPDR group, and 94% (16/17) of the moderate-severe NPDR group had an abnormal finding on at least one of the measures. Subtraction of the final column  from 100% provides the percentage of patients within each group who had some measurable abnormality for a given condition. For example, 35% (6/17) of the no NPDR group, 63% (10/16) of the mild NPDR group, and 88% (15/17) of the moderate-severe NPDR group had an abnormal finding under the melanopsin condition alone (i.e. melanopsin-mediated PLR and/or dark-adapted baseline). This finding highlights the potential usefulness of measures performed under rod- and cone-mediated conditions in addition to the more commonly used melanopsin-mediated condition, particularly for diabetic patients who had no NPDR. For these patients, the use of all three measures identified an abnormality in 71% of the sample, whereas the use of the melanopsin condition alone identified an abnormality in 35% of the sample.A summary of the baseline and PLR abnormalities is provided in The purpose of this study was to determine the effects of diabetic retinopathy on the pupil response measured under conditions designed to target selectively the rod, cone, and melanopsin pathways. Although previous work has shown abnormalities in the light- and dark-adapted steady-state pupil size in individuals who have diabetes5736There is evidence that abnormal sympathetic nervous system innervation might play a role in steady-state pupil abnormalities, but there is also evidence that the steady-state pupil size is significantly constricted in diabetics who have preserved sympathetic innervation of the iriset al.Interestingly, the melanopsin-mediated PLR (inferred ipRGC function) was not selectively spared in our diabetic patients, contrary to previous reports of selective ipRGC resistance to injury41144345The abnormally constricted steady-state pupil diameters should be considered in the interpretation of the chromatic PLR abnormalities, since a reduced steady-state pupil diameter decreases the amount of light entering the eye. The effects of reduced retinal illuminance can be estimated based on previous reports that quantified the relationship between stimulus luminance and the PLR amplitude15An additional consideration is that a substantial percentage of patients who had mild or moderate-severe NPDR had a history of treatment with anti-VEGF agents and/or focal laser therapy. However, the effects of these treatments on the PLR and the steady-state pupil size are likely minimal, as shown by the sub-analyses in 48In summary, we provide evidence that a short-duration, clinically applicable, chromatic pupillometry test can be used to assess neural dysfunction associated with diabetes. Importantly, the results suggest both inner- and outer-retinal dysfunction in some diabetic patients, indicating that pupillometry may be capable of quantifying neural abnormalities in these individuals. Future longitudinal work is needed to evaluate the extent to which pupillometry is useful for identifying individuals at risk of developing severe vascular abnormalities and vision loss.How to cite this article: Park, J. C. et al. Pupillary responses in non-proliferative diabetic retinopathy. Sci. Rep.7, 44987; doi: 10.1038/srep44987 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


We have previously reported that induced pluripotent stem cell (iPSC)-mesenchymal stem cells (MSCs) alleviated asthma inflammation in mice. Long noncoding RNAs (lncRNAs) were recently reported as being involved in the immune responses. However, whether lncRNAs are associated with iPSC-MSC immunomodulation in allergic inflammation is still unclear.Mice were induced into an asthmatic state and received treatment consisting of iPSC-MSCs. Memory T cells isolated from sensitized mice were challenged and co-cultured with iPSC-MSCs in vitro. Total RNA from the lungs and separated T cells were processed with an lncRNA/mRNA microarray. A series of bioinformatics technologies were used to screen the target lncRNAs.MM9LINCRNAEXON12105+ and AK089315 were finally emphasized via quantitative real-time PCR validation.iPSC-MSCs significantly prevented asthma inflammation and decreased the Th2 cytokine levels. Over 1300 lncRNAs were differentially expressed after the induction of asthma, and 846 or 4176 lncRNAs were differentially expressed with iPSC-MSC treatment in mice or in vitro, respectively. After overlapping the differentially expressed lncRNAs produced in a similar manner in mice and in vitro, 23 lncRNAs and 96 mRNAs were selected, in which 58 protein-coding genes were predicted to be potential targets of the 23 lncRNAs. Furthermore, using a series of bioinformatics technologies, 9 lncRNAs co-expressed with the most differentially expressed mRNAs, which were enriched in terms of the immune response, were screened out via Pearson’s correlation coefficient with mRNAs that were involved with inflammatory cytokines and receptors. lncRNAs Our results suggested that aberrant lncRNA profiles were present after asthma induction and iPSC-MSC treatment, suggesting potential targets of allergic inflammation and iPSC-MSC-mediated immunomodulation.The online version of this article (doi:10.1186/s13287-016-0456-3) contains supplementary material, which is available to authorized users. Asthma has become a global public health problem that negatively affects the quality of life of patients and causes a substantial medical and financial burden . As an a+ T-cell development and the activation process [+ T cells of severe asthma patients [Long noncoding RNAs (lncRNAs) are a group of functional RNA molecules with transcription lengths of more than 200 nucleotides which do not encode any protein products . lncRNAs process . The exppatients , 12. TheMesenchymal stem cells (MSCs) have shown broad immunomodulatory properties , 14. BonIn this study, we induced a mouse model of asthma in which iPSC-MSCs were transplanted into the mice to study the lncRNAs involved in the allergy and MSC immunomodulatory process. Moreover, an in vitro model mimicking an allergy environment was also used. Both in vivo and in vitro studies identified differentially expressed lncRNAs that might be associated with the process of airway allergy and iPSC-MSC treatment. The identified lncRNAs were screened out using a series of bioinformatics technologies and validated by quantitative real-time polymerase chain reaction (qRT-PCR). Taken together, our results provide a key foundation for lncRNA functional studies in the fields of airway allergic inflammation and iPSC-MSC immunomodulatory properties. This is the first study that links lncRNAs with the effects of iPSC-MSCs in allergic airway inflammation.Female BALB/c mice (4–6 weeks of age) were purchased from the Guangdong Medical Laboratory Animal Centre . All procedures were performed according to the protocols approved by the Sun Yat-sen University Institutional Animal Care and Use Committee.Effects of mesenchymal stem cells from human iPS cells on differentiation, maturation, and function of dendritic cells, which is under revision in Stem Cell Research & Therapy. Similar to general MSCs, iPSC-MSCs were positive for CD105, CD73, CD90, CD146, CD144, and CD44, and negative for CD34, CD14, and CD45. iPSC-MSC activities of osteogenic, chondrogenic, and adipogenic differentiation were confirmed. Detailed information on iPSC-MSCs is presented in Additional file The human iPSC-MSCs used in this study were prepared in a similar way to our previous study, titled n = 15, 16, and 15 for phosphate-buffered saline (PBS)/PBS/PBS, ovalbumin (OVA)/OVA/PBS, and OVA/OVA/iPSC-MSC, respectively). Airway responsiveness to methacholine (Mch), asthmatic inflammation in the lung, and RNA microarray extraction were analyzed. The details are presented in Additional file The mouse model of asthma was established according to our previous description with minor modifications , 19. Then = 8) were sensitized by OVA on day 1 and day 7 and were sacrificed on day 14 to obtain the memory T (Tm) cells [The mice (m) cells . The Tm n = 3 for each group), total RNA for the lncRNA and mRNA microarrays was extracted from the lung tissues of three different groups . For the in vitro study (n = 3 for each group), total RNA for the lncRNA and mRNA microarrays was extracted from Tm cells that separated from the co-culture of two different groups (Tm + OVA and Tm + OVA + iPSC-MSC). The microarray service was provided by Arraystar . The extract data were processed using the Agilent Feature Extraction Software.For the in vivo study  within 10 kb of the known genes. For trans-acting analysis, we used BLAST software for the first round of screening (e < 1E-20) and the RNAplex software to choose trans-acting targets. The RNAplex parameters were set as –e-30.p < 0.05 was considered correlative.A Pearson’s correlation coefficient between the lncRNAs and mRNAs associated with the immune response was constructed. A Pearson’s correlation coefficient >0.6 or < –0.6 and A gene ontology (GO) analysis was performed to characterize genes and gene products in terms of the biological process (BP), cellular component (CC), and molecular function (MF). Fisher’s exact test was used to find if there was overlap between the differentially expressed list and the GO annotation list. The pathway analysis was a functional analysis that maps genes to KEGG pathways. This analysis was used to determine the main pathways in which differentially expressed mRNAs were enriched.n = 6, 7, and 6 for PBS/PBS/PBS, OVA/OVA/PBS, and OVA/OVA/iPSC-MSC, respectively). The details are presented in Additional file Aberrant lncRNAs were finally selected from the lncRNA microarray results and were confirmed with additional samples using qRT-PCR  followed by post-hoc Tukey  or Dunnett T3  tests were used for multiple comparisons between different groups. A Kruskal–Wallis rank sum test followed by a Mann–Whitney Figure Similar to our previous study , 19, thep < 0.01; Fig. p < 0.01; Fig. To further identify the effects of iPSC-MSCs on Th2 responses and to identify the possible lncRNAs involved in the immunomodulation of iPSC-MSCs from the huge amount of microarray data, we used an in vitro model to mimic the Th2 environment. The Tm cells were isolated and purified from the spleen mononuclear cells of mice, which were sensitized twice using OVA and then were further stimulated with OVA in culture systems. We found that both IL-4 and IL-13 Fig. , but notp < 0.05 and fold change >2).mRNA and lncRNA expression profiles were measured via microarrays. The lncRNA microarray covered more than 24,000 lncRNAs in mice. As shown in Fig. The key lncRNA regulators that presented the reverse variation trends between asthma induction and iPSC-MSC transplantation should have more significance for our exploration of the possible mechanisms of MSC-mediated immunomodulation. Therefore, we next selected two patterns with opposite directions (up then down or down then up) after the asthma induction and after iPSC-MSC treatment for further study Fig. . HoweverSimilarly, we found that a total of 88 mRNAs were differentially expressed with a pattern of up then down in mice and down in vitro Fig. . Eight mThe pathogenesis of allergic inflammation is a complex progress associated with various immunocyte dysfunctions, including T lymphocytes, ILCs, DCs, etc. To confirm the expression patterns of lncRNAs and mRNAs associated with allergy and iPSC-MSC immunomodulation comprehensively, hierarchical clustering was performed to analyze the expressions of 213 lncRNAs and 307 mRNAs in vivo. The data showed a distinguishable lncRNA and mRNA expression profiling pattern between the OVA/OVA/PBS group and the OVA/OVA/iPSC-MSC group Fig. . The heaTo predict the potential targets of differentially expressed lncRNAs involved in allergy and iPSC-MSC immunomodulation, cis and trans analyses of the selected 23 lncRNAs were performed. A cis-acting analysis was used to predict the targets of lncRNAs; a major function of lncRNAs is regulating the neighboring mRNA expression through cis-acting mechanisms. For the cis-acting analysis, the genomic position of the selected 23 lncRNAs relative to the genomic positions of their closest known genes were determined from the RefSeq and UCSC Known Genes databases . We searIl4, Il5, Il13, Il10, Jak1, Jak3, Stat6, Gata3, Foxp3, Ccl17, and Ccl22 which related to inflammatory cytokines and receptors, were chosen to confirm their relationships with the 23 selected lncRNAs. Each of the selected lncRNAs was used to calculate the Pearson’s correlation coefficient and p value with 72 mRNAs; nine out of 23 lncRNAs that had more than 40 co-expressed mRNAs were screened out .To further identify the lncRNAs that were closely connected to an immune response, a correlation analysis of 23 selected lncRNAs and mRNAs involved in allergic inflammation and iPSC-MSC immunomodulation was carried out. Seventy-two mRNAs, including p value <0.05). These lncRNAs had a co-expression with most of the 96 mRNAs; a pattern of high numbers of connections between protein-coding mRNAs and lncRNAs is also observed in a visual solution . Another was completely the opposite (down then up in mice and up in vitro). Twenty-three lncRNAs were screened out via the overlap of the in vivo study and in vitro study. Furthermore, nine out of 23 lncRNAs were identified as being involved in the immune response after the analysis of the target prediction, GO, and pathway analysis, and Pearson’s correlation coefficient.Our microarray analysis identified that a total of 213 lncRNAs were differentially expressed in opposite directions (down then up or up then down) after the asthma induction and after iPSC-MSC treatment. However, the number of candidate lncRNAs was still too large. An in vitro study was supplemented to narrow the scope. IL-4 and IL-13, but not IL-5, were highly secreted by the sensitized Tm cells after challenges in the culture. These results prove that we utilized a proper in vitro model for allergy research. Moreover, for the first time ever, we demonstrated that iPSC-MSCs dramatically decreased the levels of IL-4 and IL-13 in Il4, Il5, and Il13 [Stat5 and Stat6 [Foxp3 [Ccl17 and Ccl22, which play a critical role in the recruitment and activation of Th2 cells [Il4, Il5, and Il13 were regulated by iPSC-MSC treatment [We also carried out an mRNA microarray at the same time. mRNAs involved in allergy and iPSC-MSC treatment were also identified, and their relationships with the lncRNAs were analyzed. Among the 72 mRNAs co-expressed with the selected 23 lncRNAs, some were closely associated with allergic inflammation, such as: Th2 cytokine coding genes, including and Il13 ; Th2-spend Stat6 ; Treg tr6 [Foxp3 ; and cheh2 cells . The proreatment , and theMM9LINCRNAEXON12105+ and AK089315, were proven to be the most relevant because they differentially expressed significant differences in the airway allergy and iPSC-MSC treatment. For further functional study, genomic modifications of the above two selected lncRNAs could be used to explore their functions in identified mRNA regulation. The targeted lncRNA knockout or overexpressed T cells that were challenged and co-cultured with iPSC-MSCs in vitro could be processed with a microarray or via RNA-seq analysis to verify that the mRNAs are regulated by a specific lncRNA. The results matched the identified mRNAs associated with allergy, and iPSC-MSC treatment should be selected. The mRNAs mediated by two selected lncRNAs would be determined, and the lncRNA/mRNA pathway involved in the allergic reaction and iPSC-MSC immunoregulation could be verified. We have attempted to perform genomic modification of the two target lncRNAs in murine primary Tm cells with small-interfering RNA. However, primary T lymphocytes are generally resistant to non-viral vector-based transfection methods [Despite accumulating evidence suggesting that the majority of lncRNAs are likely to be functional , 37, we  methods . SignifiIn our study, the in vitro study was supplemented to help narrow the scope of lncRNAs selected from the in vivo data. The advantage of this was effective in focusing on the target lncRNAs. However, the limitation was that the samples between the in vivo and in vitro study were different. The lungs used for the in vivo study not only contained T lymphocytes, which were used for the in vitro study, but also included other types of cells such as epithelial cells, ILC2, and DCs. Therefore, the selected lncRNAs in this study still focused on T lymphocytes. The selected lncRNAs involved in the allergy and iPSC-MSC immunomodulation may also be associated with other types of cells, such as epithelial cells and DCs. Moreover, MSCs were also shown to regulate epithelial cells, DCs, etc. , 40, whiIn conclusion, our study found that airway allergic inflammation can cause alterations of lncRNAs expressed in mice, and some of these altered lncRNAs are also involved in the alleviation of iPSC-MSC airway inflammation. This study evaluated the aberrant lncRNA expression profiles both in asthma and iPSC-MSC treatment, which demonstrated that lncRNAs may be related to allergy and iPSC-MSC immunomodulation. This study provides a key foundation to study the mechanisms underlying airway allergic inflammation and iPSC-MSC immunomodulation, which may be helpful in promoting iPSC-MSC medical applications.


Verrucomicrobia contains freshwater representatives which remain poorly studied at the genomic, taxonomic, and ecological levels. In this work we present eighteen new reconstructed verrucomicrobial genomes from two freshwater reservoirs located close to each other . These metagenome-assembled genomes (MAGs) display a remarkable taxonomic diversity inside the phylum and comprise wide ranges of estimated genome sizes (from 1.8 to 6 Mb). Among all Verrucomicrobia studied we found some of the smallest genomes of the Spartobacteria and Opitutae classes described so far. Some of the Opitutae family MAGs were small, cosmopolitan, with a general heterotrophic metabolism with preference for carbohydrates, and capable of xylan, chitin, or cellulose degradation. Besides, we assembled large copiotroph genomes, which contain a higher number of transporters, polysaccharide degrading pathways and in general more strategies for the uptake of nutrients and carbohydrate-based metabolic pathways in comparison with the representatives with the smaller genomes. The diverse genomes revealed interesting features like green-light absorbing rhodopsins and a complete set of genes involved in nitrogen fixation. The large diversity in genome sizes and physiological properties emphasize the diversity of this clade in freshwaters enlarging even further the already broad eco-physiological range of these microbes.The phylum  Verrucomicrobia were originally classified by a largely morphology-based taxonomic approach into a broad taxon called the “Caulobacterales,” a diverse group of microbes bearing “prosthecae”  experiments targeting specific Verrucomicrobia clades in humic lakes have shown that freshwater Verrucomicrobia are diverse and may have large populations up to ca. 19% of the total community  inferred to be polysaccharide-degraders have been sequenced from the marine environment (Verrucomicrobia bacterium Ga0077533) are available. Only a single SAG has been described from the freshwater Lake Mendota  located in Southeast Spain. The large number of verrucomicrobial genomes (18) reconstructed from these freshwater reservoirs show broad differences in genomic make up within the same verrucomicrobial class Opitutae, likely reflecting specialization in different ecological strategies (oligotrophic K-strategists versus copiotrophic r-strategists). The data presented contribute a large number of novel verrucomicrobial groups detected for the first time through these MAGs.Here we have used metagenomic tools to reconstruct novel freshwater A total of five datasets from two different Spanish freshwater reservoirs were used in this work. Four of them have already appeared in publications and came from winter season, when the water column was mixed. Two from Amadorio reservoir  and anote-value < 1e-5 it was considered a potential 16S rRNA gene fragment. These candidate reads were aligned to archaeal, bacterial, and eukaryal 16S/18S rRNA HMM models using ssu-align to identify true 16S/18S sequences  into approximately 800,000 sequences at 90% identity level using UCLUST . This daequences . The finVerrucomicrobia MAGs from Amadorio were reconstructed using the small and large fractions of Amadorio reservoir datasets  to this phylum were obtained for >60% of the total genes presented in each contig. More specifically, contigs falling within these parameters were binned using taxonomy, principal component analysis of tetranucleotide frequencies, GC content, and coverage values in Tous and Amadorio metagenomes  with >50% of genome completeness assessed by CheckM . RPKG is a normalized number comparable across metagenomes from different locations. All metagenomic datasets used in this work are publicly available: Amadorio  . The FISH protocol was performed as previously described  . Once in5% (w/v)  (SIGMA) 5% (w/v) . Water smicrobia . FISH-spER3 tool  from speER3 tool , RDP Relescribed . HybridiVerrucomicrobia was particularly high, reaching 20% of the total 16S rRNA reads of these datasets. No correlation of verrucomicrobial abundances was apparent either with latitude or with trophic status, although it is evident that they are found in all kinds of freshwater lakes or reservoirs.We have estimated the relative abundance of verrucomicrobial 16S rRNA in several available freshwater metagenomic datasets, ranging from oligotrophic to eutrophic and tropical to cold, distributed in different latitudes and altitudes. We have found a relative abundance between 2 and 19%  we were able to reconstruct a total of 18 Verrucomicrobia genomes (Table 1) of which 15 were different genomes as suggested by ANI calculations. Only three pairs of genomes displayed an ANI higher than 98% suggesting they came from the same microbe . Some of these genomes appear to be the smallest verrucomicrobial representatives described yet , Spartobacteria (subdivision 2), and Verrucomicrobiae (subdivision 1). There are also two subdivisions (6 and 7) which comprise unclassified representatives and another one comprising Pedosphaera parvula like microbes (subdivision 3). Recently, one divergent member from the verrucomicrobial subdivision 5 has been described as a novel phylum-level lineage , firstly small verrucomicrobial genomes, with relatively smaller intergenic spacers compared to those seen in reference marine , freshwater , or brackish genomes . These small genomes belong to the class Opitutae within the family Puniceicoccales  and the class Spartobacteria . They have estimated genome sizes of 1.83–2.27 Mb, with median intergenic spacers smaller than 38 bp , reaching in some cases 5 Mb (Pedosphaera-Tous-C6FEB), which exhibit larger genome sizes, although still smaller than the 6–8 Mb seen in soil representatives , symbionts of termites  or associated to sugar beet plants . Only a single genome was estimated to have a size >6 Mb .The genome sizes estimated from these MAGs suggest we have obtained two types of microbes (Actinobacteria or LD12 Alphaproteobacteria) to relatively large genomes of r-strategists like Limnohabitans . It is evident how the genomes presented in this work cover the whole range of streamlining from one end to the other. Overall, we have obtained a great diversity of genomes from all known Verrucomicrobia classes, although we were able to reconstruct more genomes belonging to the class Opitutae. We have focused our efforts in a comparative study between the two kinds of Opitutae genomes, that appear to correspond also to a separation between endemic and cosmopolitan microbes (see below).The verrucomicrobial genomes have been plotted by estimated genome sizes and median intergenic spacers (as indicators of how streamlined the genomes are) together with other typical freshwater microbes that go from the very streamlined K-strategists  reconstructed Verrucomicrobia  and the specific groups (Verrucomicrobia-Tous-C9LFEB for Tous and Opitutae-AMD-G3 for Amadorio) in the vertical profile (samples were taken every 2 m depth and fixed for microscopy) under summer stratification conditions was addressed by FISH in both reservoirs . The distribution pattern in the vertical profile of the percentage of total Verrucomicrobia with respect to the total heterotrophs was similar in both reservoirs, with their relative contribution increasing with depth. In both cases the lowest values were observed in the upper waters (epilimnion) with an average of 2.5% in Tous and a 2.8% in Amadorio, gradually increasing toward the bottom of the reservoirs with deep maxima of Verrucomicrobia contribution to total bacterioplankton of ca. 6% in Tous and 7% in Amadorio. FISH counts using the VrmC9LTo-specific probe of Verrucomicrobia-Tous-C9LFEB show that this group represents only a small part of the total heterotrophs of Tous Reservoir during summer stratification, always below 1%, with a relatively constant contribution of ca. 15% of total Verrucomicrobia within the water column. The VrmG3Ama probe, which is specific of Opitutae-AMD-G3 from Amadorio reservoir, allowed us determining that this group was present at a relatively low contribution in relation to total heterotrophs (<1%), however, from 12 m depth to the bottom its abundance increased to 3–4% with respect to the total of heterotrophs and contributed over 50% of the total Verrucomicrobia. These data confirm that Opitutae-AMD-G3 (2.13 Mb of estimated genome size) is a very abundant microbe in Amadorio , which fits with the proposal that these microbes are K-strategists, while Verrucomicrobia-Tous-C9LFEB (large genome of 4.63 Mb of estimated genome size) contributed at very low numbers in Tous , which fits with the idea that it possesses an opportunistic strategy.The distribution of total planktonic Verrucomicrobia appear to be involved in the metabolism of two glycosphingolipids, galactosylceramides, and sulfatides, a pathway that is typically found in eukaryotes, presenting several enzymes like aryl-sulfatases, sialidases, and beta-galactosidases. Among the diverse microbes reconstructed in this work, these features were widespread and common to the majority of them, although certain pathways for mono and polysaccharide utilization were specific to some (see below). Despite the general aerobic and heterotrophic metabolism inferred, we were able to identify some microbes with anaerobic reductase complexes (Pedosphaera-Tous-C6FEB), some of them could be able to perform photoheterotrophic lifestyles through rhodopsin pumps and one of them could be capable of nitrogen fixation.In general, we found an aerobic heterotrophic metabolism with degradation of polysaccharides in all of our genomes, containing metabolic pathways involved in glycolate, glucarate/galactorate, glucoronate/galacturonate, gluconate, polysaccharides, monosaccharides, and aminosugars utilization and degradation. We also noticed a large amount of Ton/Tol/Exb receptors for transport of polymers, vitamins, and iron uptake, and carbon storage preference for glycogen (which is both synthesized and degraded) instead of poly-[(beta)]-hydroxybutyrate (PHB). Interestingly, Verrucomicrobia are known to be able to tackle more complex and recalcitrant polysaccharides . We also detected some other abundant GHs families as GH33 , GH74 (endoglucanase and xyloglucanase) and GH2 , GH13_9 (α-amylase), and GH77 . In general, we detected a growing proportion of GHs with the increase in MAG genome size and the total number of CDS. It also correlates with a higher number of transporters and metabolic pathways found in the larger genomes compared to the small genomes (see below). It is noteworthy that Verrucomicrobia-Tous-C9LFEB contained the highest number of GHs (218), a huge number compared to the other MAGs, which contained between 2 and 80 GHs. This microbe, as explained below, contains the highest and most remarkable variety of metabolic pathways of all the genomes compared here, including nitrogen fixation and efficient degradation of recalcitrant compounds such as chitin, cellulose, xylan, starch, or mannan, among others.In addition to more simple sugars, charides . We perfcharides  and dbCAcharides  databaseFigure 4), which have genome size estimations of around 2.2 Mb, being among the smallest Verrucomicrobia representatives ever found, at least in freshwater ecosystems. We also studied more in depth two of the largest genomes that appear sporadically in metagenomes (see above), Opitutae-Tous-C4FEB  and Verrucomicrobia-Tous-C9LFEB  were common to both small and large genomes, although glycogen debranching enzyme (glgX) was only detected in the small Opitutae-AMD-G1. A specific operon (oleABCD) that has been previously described for long chain alkene biosynthesis from acyl-CoA in some members of Planctomycetes, Verrucomicrobia, Actinobacteria, Chloroflexi, and Betaproteobacteria  was their ability to effectively degrade different types of mono and polysaccharides (as described above). Sialidases , which release N-acetylneuraminate residues from glycans, glycoproteins, and polysaccharides (N-acetyl-D-glucosamine (chitin) degradation in small Opitutae-AMD-G1 and big Opitutae-Tous-C4FEB . Complete pathways with GHs for mannose and rhamnose utilization were only detected in the unclassified Verrucomicrobia-Tous-C9LFEB. The two large genomes were able to utilize sugar alcohol carbon sources, since they have a glycerol-3-phosphate transporter and a glycerol-3-phosphate dehydrogenase. Pathways for inositol catabolism to form glucuronate and transformation of the latter to glyceraldehyde-3-phosphate, as well as fructose utilization via PTS transport system were also common in these large genomes.One of the most remarkable features of these freshwater charides  were fouGenerally, we detected a high number of Exb, Ton, and Tol biopolymer and iron transporters in all the reconstructed genomes, although we also noticed an increase in number of these transporter with increasing genome size. The presence of these transporters suggests an alternative route for incorporation and recycling of dissolved organic matter in freshwater bodies, which was already described in marine ecosystems . In geneVerrucomicrobia, like freshwater Actinobacteria, are important in inorganic phosphorous sequestering which could contribute to prevent eutrophication , alkaline phosphatase, or inorganic phosphate transporters, which suggest that hication . The stocum SolV . Polyamiomycetes ; only smBacterial nitric oxide synthases have been described in the past years as enzymes involved in toxin biosynthesis, protection against oxidative stress, and regulation of recovery from radiation damage . In OpitVerrucomicrobia. For instance, Opitutae-Tous-C4 , a 3.9 Mb microbe (estimated genome size) contains almost all of the pathways described in the small Opitutae (see above) plus other pathways suggesting a photoheterotrophic metabolism, including a complete pathway for carotenoids biosynthesis together with a proteorhodopsin proton-pump. Carbonic anhydrases, phosphoenolpyruvate carboxylase, or PhrB photolyases for stress response to light were also detected. Compared to the small genomes, we found more vitamin  and cofactor biosynthetic pathways, including one vitamin B12 transporter in Opitutae-Tous-C4.We found a cluster of flagellum genes for motility functions exclusively in the large genomes (Opitutae-Tous-C4FEB and Verrucomicrobia-Tous-C9LFEB), which was totally absent in the rest of the reconstructed genomes of this work. A CRISPR-Cas type I-C was also found in Opitutae-Tous-C4FEB. Some of the main differences between small and large genomes were based on their ability to perform other different types of metabolism which couple with the general heterotrophic metabolism in Verrucomicrobia, including methanotrophs and termite symbionts (Verrucomicrobia (Verrucomicrobia-Tous-C9LFEB) for nitrogen fixation. Typically, a minimum set of six nif genes is required in bacteria for nitrogen fixation, three of them code for structural and catalytic components (NifHDK) and the other three for biosynthetic proteins (NifENB)  . We founVerrucomicrobia nitrogenases which have representatives in termite guts (O. bacterium TAV5 and TSB47), soil (Terrimicrobium sacchariphilum), and sea (C. akajimensiis). Similar to what can be seen for the entire operon  these nitrogenases are more divergent compared to the verrucomicrobial Methylacidiphilum representatives . Another peculiar feature of the operon is the apparent insertion/translocation of the gene encoding the pII nitrogen regulatory protein, between nifD and nifH genes. Moreover, the pII gene also appears to be duplicated.A comparison of the Cyanobacteria . Additionally, Verrucomicrobiae-Tous-C5FEB contains one novel rhodopsin belonging to a new clade of rhodopsin proton pumps, its closest sequences being the rhodopsins from Exiguobacterium . This great size variability of reconstructed genomes, ranging from about 2 to 6 Mb, would shed light onto the classification inside the phylum. We found a correlation between both ends of the genome size spectrum and the distribution in diverse freshwater and brackish metagenomes around the world with some small genomes being extremely widespread and abundant and others, particularly the large genomes, with much more restricted distribution. Although all the genomes seem to correspond to aerobic heterotrophs degrading polysaccharides, the larger genomes display much more transport potential and have likely more diversified metabolism. There is a group that appears to be adapted to the stratified period at DCM depths while the majority of genomes appear to be more abundant in deeper waters  not typical from freshwater well-oxygenated systems like Tous reservoir, which never undergoes anoxic conditions. The photoheterotrophic metabolism in some of the microbes presented here was confirmed with the discovery of freshwater Planctomycetes–Verrucomicrobia rhodopsins, likely a novel branch within this already hyperdiversified family of proteins. It is noteworthy that the small Opitutae-AMD-G3 and Opitutae-AMD-G1 described here are cosmopolitan in lakes from different environmental conditions, ranging from cold oligotrophic lakes , temperate oligotrophic (Lake Lanier or Tous reservoir), and temperate mesotrophic (Amadorio) or eutrophic (Lake Mendota). We have visualized by FISH the microbes represented by Opitutae-AMD-G3 and Verrucomicrobia-Tous-C9LFEB and would like to propose Candidatus names for both microbes: Candidatus Opitutus lacustris AMD-G3 and Candidatus Verrucococcus diazotrophicus Tous-C9LFEB.We found a nitrogen fixing cluster, a characteristic never described before in free living freshwater PRJNA342151 . Amadorio reservoir metagenomic datasets are available under BioProject number PRJNA238866. The 18 assembled Verrucomicrobia genomes have been deposited in the NCBI under Biosample identifiers SAMN06820742-SAMN06820759.Tous reservoir metagenomic datasets have been deposited in the NCBI SRA database with BioProject number RG, FR-V, and PC-Y conceived this work. PC-Y and AP performed the sample collection and filtration. PC-Y performed the DNA extraction. Analysis was carried out by PC-Y, RG, MM, and AP. Manuscript was written by PC-Y, FR-V, and RG. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Circulating microvesicles (MVs) are suggested to be important contributors to cancer-associated thrombosis due to the presence of surface-bound procoagulant molecules like tissue factor (TF) and phosphatidylserine (PS). Pancreatic cancer is considered to be one of the most prothrombotic malignancies. The aim of this study was to describe the impact of analytical variables on MV-associated thrombin generation in patients with pancreatic cancer and in healthy controls. MVs were isolated from citrated plasma and added to pooled normal plasma (PNP). Thrombin generation was measured by the calibrated automated thrombogram. The impact of corn trypsin inhibitor (CTI), anti-tissue factor pathway inhibitor (TFPI) antibodies and phospholipids was described. Antibodies against TF were used to assess TF-dependency, and MV-bound PS activity was measured with the Zymuphen MP-activity kit. MVs from the pancreatic cancer patients displayed higher thrombin generation and higher PS-activity than MVs from the healthy control group, while TF-dependency was observed in only 1 out of 13 patient samples. Adequate thrombin generation-curves were only achieved when CTI was omitted and anti-TFPI antibodies were added to PNP prepared in low contact-activating tubes. Addition of phospholipids reduced the significant differences between the two groups, and should be omitted. This modified thrombin generation assay could be useful for measurement of procoagulant circulating MVs, allowing the contribution from MVs affecting both the intrinsic and the extrinsic pathway to be measured. Cancer patients are at increased risk of developing venous thromboembolism (VTE), and growing evidence suggests that circulating microvesicles (MVs) are important contributors to the prothrombotic state , 2. MVs et al. for measuring thrombin generation in platelet-rich or platelet-poor plasma in a variety of clinical settings [At present, the reports of PS/TF-positive MVs as biomarkers for VTE in cancer are inconsistent, to which methodological limitations and lack of standardization probably contribute considerably , 19–21. settings . In a resettings . A modifsettings –27. In ssettings .Other ways to modulate the thrombin generation is to target plasma inhibitors , 30. TisThe aim of this study was to describe the effect of CTI, anti-TFPI abs and phospholipids (MP-reagent) on the MV-associated thrombin generation in samples from patients with pancreatic cancer and healthy controls, and find the best conditions to compare these two groups.The project was approved by the Regional Committee for Medical and Health Research Ethics, South-Eastern Norway (2015/1124/REK sør-øst C) and by the Data Protection Supervisor at Oslo University Hospital (2015/9517). The patients were originally included in the Thematic Pancreatic Tumour Project (TPTP), which was approved by the Regional Committee for Medical and Health Research Ethics, South-Eastern Norway (265–08412 C), the Norwegian Directorate of Health (08/7927) and The Norwegian Data Protection Authority (08/01409-2).st 2012 and September 30th 2013. Citrated plasma samples were available for 14 of these patients. The inclusion criteria for patients in the present study was newly diagnosed and pathologically confirmed (cytology and/or histology) pancreatic cancer and absence of another previous (the last five years) cancer diagnosis. Among the 14 patients one was excluded due to concomitant diagnosis of another cancer. The group included eight men and five women and the mean age at diagnosis was 64.1 years (SD 7.3). Among the 13 patients, four used acetylsalicylic acid (as prophylaxis of cardiovascular disease) and one used warfarin due to several previous venous thromboembolisms. None of the patients had started with chemotherapy or used hormone therapy. The controls (n = 13) were recruited, in retrospect, among healthy candidates that matched the patients by age (+/- five years) and gender. None of the controls used anticoagulants. All study subjects (patients and controls) gave their written informed consent, and blood was drawn without requirements of fasting. Patient plasmas were collected in 2012–2013 and plasmas from healthy controls were collected in 2015. Citrate-anticoagulated blood  was collected by venipuncture (21G needle) and the first tube was discarded. Plasma was prepared at 2,500 g, 15 min, room temperature (RT) within one hour after blood collection, and then aliquoted and stored frozen at -80°.The patients were originally included prospectively in the Thematic Pancreatic Tumour Project (TPTP) between January 1Tris buffered saline (TBS) and bovine serum albumin (BSA) were purchased from Sigma Aldrich . Thrombin Calibrator, MP-reagent (4 μM phospholipids) and FluCa-kit were from Thrombinoscope BV . The anti-human TFPI abs (CLB/TFPI C-terminus) was from Sanquin Reagents . CTI was purchased from Enzyme Research Laboratories . The hybridoma cells TF8-5G9 were a kind gift from professor James H.Morrissey , and The Core Facility for monoclonal antibody production and assay design  produced the TF8-5G9 anti-TF abs.Citrated plasma (>500 μL) from pancreatic cancer patients and healthy controls was thawed (once) for 15 min at 37°C, and then 500 μL plasma was transferred to a new tube and spun to pellet the MVs . Subsequently, 450 μL plasma was removed, and the remaining MV-enriched pellet was washed with 450 μL tris-buffered saline with 0.5% v/v bovine serum albumin (TBSA), briefly vortexed and centrifuged again . The supernatant was removed, and the MV pellet was dissolved in 50 μL TBSA and vortexed for 5 sec.in vitro thrombin generation experiments, where 100 μg/mL of anti-TFPI abs gave the highest sensitivity for detection of MVs obtained from whole blood stimulated with Neisseria meningitidis bacteria.Blood was collected in Monovette tubes  with and without manually prefilled CTI . The venipuncture was performed with a Safety-Multifly®-Needle 21G , and the first tube was discarded. After written informed consent, eight healthy volunteers donated blood to four tubes with CTI and four tubes without CTI. The tubes were centrifuged  and pooled into two separate plasmas (+/- CTI) which were subjected to another centrifugation at 2,000 g, 15 min, RT. The PNP (+/- CTI) was aliquoted and stored at -80°C. At the day of use, the PNP (+/- CTI) was thawed at RT, and incubated (15 min at 37°) with anti-human TFPI abs  or an equal volume of TBSA before addition to the wells. The concentration of anti-TFPI abs was chosen based on initial et al [The ability of plasma-derived MVs to generate thrombin was measured with a modified version of the CAT originally described by Hemker et al . Seventyet al . Of noteCitrated plasma from pancreatic cancer patients and healthy controls was thawed (for a second time) at 37°C for 15 min. The activity of phosphatidylserine (PS) in the plasma (1:50 dilution) was measured with the Zymuphen MP-activity assay . Briefly, PS-positive MVs are captured on an ELISA plate coated with annexinV-streptavidin. In the presence of FVa/FXa and calcium, captured MVs with negatively charged phospholipids cleave prothrombin to thrombin, and the phospholipid concentration is the rate-limiting step. Finally, a chromogenic substrate for thrombin is added to assess the levels of thrombin. The results are expressed as PS-equivalents (nM).All results are presented as mean (SD), and comparison of means were performed with repeated measures (RM) two-way ANOVA with Sidak’s multiple comparison test, or an unpaired t-test with Welch correction . Associations between thrombin generation parameters and PS equivalents were evaluated with Pearson’s test. All tests were performed with GraphPad Prism version 6.01. Repeatability is reported as coefficient of variation (CV) from duplicate measurements, made across a number of different experiments and subjects .The PNP (with 18.3 μg/mL CTI) itself did not generate any thrombin during 90 min of recording. MVs isolated from plasma from patients with pancreatic cancer and healthy controls generated small amounts of thrombin when added to this PNP . Most ofAddition of anti-TFPI abs shortened the LTs significantly in both the pancreatic cancer and the healthy control group, and it also reduced the variability between individuals in both groups, most evidently within the healthy group . The CV Neisseria meningitidis (108/mL) for 4 hours served as positive controls (n = 6), and these samples were analyzed in the same run as the patient samples. Consistent across the four assay conditions, the positive controls showed an evident prolongation of the LT after preincubation with anti-TF abs  itself did not generate any thrombin during 90 min of recording. In this PNP, native thrombin generation of MVs from the control group was comparable to what we observed in PNP with CTI, with most of the curves not reaching the baseline  nM thrombin x min (p<0.0001), peak: 191 (48) vs 89 (23) nM thrombin (p<0.0001) and velocity index: 53 (27) vs 15 (6) nM thrombin min (p = 0.0001) , 1.5% (ETP), 6% (peak) and 15% (velocity index)  would permit detection of TF-dependent thrombin generation. However, in initial experiments, using PNP with CTI, we only detected a clear TF-dependency in 1/13 samples from the pancreatic cancer patients . Also, wThe activity of PS-exposing MVs was significantly higher in plasma from patients with pancreatic cancer than in healthy controls, with a mean (SD) of 57 (44) and 7 (2) nM PS equivalents, respectively .in vitro [et al have reported that in vivo-generated MVs from platelets and erythrocytes can initiate and propagate thrombin independently of TF in a FXII-dependent manner [et al showed that extracellular vesicle (EV)-associated poly-phosphates (poly-P) in plasma from prostate cancer patients initiate coagulation in a FXII-dependent manner [In this study, we present the impact of the analytical variables CTI, anti-TFPI-abs and phospholipids (MP-reagent) on MV-associated thrombin generation in patients with pancreatic cancer and in healthy controls. PNP prepared without CTI in blood collection tubes with low contact activation, and inhibiting abs against TFPI added, offered the best assay conditions to measure MV-associated thrombin generation in the two groups. In our study, most of the MV-samples generated too low amounts of thrombin during native conditions (i.e. measurements without the addition of an MP-reagent or anti-TFPI abs). The thrombin generation curves did not reach the baseline, which made the LT the only objective parameter to evaluate. Furthermore, native MV-associated thrombin generation also gave numerous LTs > 20 min, where the duplicate CVs were relatively high. Others have reported anti-TFPI antibodies to increase TF-dependent thrombin generation of MVs generated in vitro , 32. Howin vitro , the usein vitro . Previout manner . Recentlt manner . They alt manner .The Monovette blood collection tubes we used for preparation of PNP have previously been reported to induce minimal contact activation compared to other commercially available tubes . In accoThe boosting effect of anti-TFPI abs was much more pronounced in the absence of CTI, which may indicate that the anti-TFPI abs allows amplification of MV-associated molecules that affects the intrinsic pathway. It is well known that TFPIα affects the extrinsic pathway by inhibiting the TF-FVIIa complex in a FXa-dependent manner , but TFPet al did not report significant differences in fibrin generation time between cancer patients and healthy individuals [et al reported that MVs from patients with metastatic pancreatic cancer had a limited effect on time to fibrin clot formation despite a significantly higher MV-associated TF-activity as measured with a chromogenic, factor Xa-based assay [vs thrombin generation. Also, during our conditions it is possible to evaluate the whole thrombin generation curve, rather than just the initiation of coagulation. Endogenous PS, as well as other MV-associated molecules such as polyphosphates, may amplify the overall thrombin generation response. Therefore, it is important to also estimate other parameters than the LT/time to the initiation of fibrin clot. Compared with healthy controls, the level of MV-associated PS was highly elevated in our pancreatic cancer group. This is in contrast to the study by Thaler et al [With our optimized assay conditions , we showed a significantly different procoagulant potential of MVs from patients with pancreatic cancer compared with MVs from healthy individuals. In comparison, van Doormaal ividuals , and Thaed assay . Howeverer et al , but in er et al , 17, 43.The contribution of TF to the MV-associated thrombin generation was analyzed in CTI-containing PNP, which should be the optimal condition for the detection of low TF. Except for in one patient with pancreatic cancer, we did not find MV-associated thrombin generation that was clearly dependent on TF across the four assay conditions. Of note, none of the patients with pancreatic cancer had clinical symptoms of disseminated intravascular coagulation (DIC) or ongoing thrombotic complications at the time point of blood collection. Other studies have reported increased MV-bound TF activity in cancer patients with thrombotic complications , 17, 44,et al recently reported that the concentration of PS-positive vesicles in plasma was not affected after one year of storage, whereas a 7-fold increase in the number of PS-exposing EVs was found after a single freeze-thaw cycle [There are limitations to our study that should be mentioned. The plasma from the pancreatic cancer patients was stored 2–3 years longer than the plasma from healthy controls. Yuana aw cycle . Our samThe most favorable analytical condition to measure MV-associated thrombin generation in patients with pancreatic cancer and healthy controls was to add the isolated MVs into low contact-activated PNP without CTI and without external phospholipids, but in the presence of anti-TFPI antibodies. These assay conditions gave good repeatability, and the contribution from MVs affecting both the extrinsic and intrinsic pathway can be measured. Although demonstrated in samples from patients with meningococcal sepsis, the ability to detect MV- associated TF-activity (by adding anti-TF abs) in diseases with low TF, remains to be elucidated. This modified thrombin generation assay could be a valuable contribution to the existing methods for measuring circulating MVs in conditions with increased risk of thrombotic complications.S1 FigMVs were preincubated with or without anti-TF abs, and analyzed in pooled normal plasma (PNP) without corn trypsin inhibitor (CTI), but with anti-TFPI abs added.(TIF)Click here for additional data file.S2 FigThe associations between the TG parameters Peak, lag time, velocity index and ETP for the pancreatic cancer patients samples are presented .(TIF)Click here for additional data file.S1 Table1 and x2) in samples from both the pancreatic cancer patients (P1-P13) and the healthy controls (HC1-HC13). In the experiments performed in PNP with CTI, the results from the samples incubated with anti-TF abs were also included (n = 52). The formulas used are:CV calculations are based on duplicate measurements (x(PDF)Click here for additional data file.