Leishmania donovani, while invading macrophages, encounters striking shift in temperature and pH (from 22°C and pH 7.2 to 37°C and pH 5.5), which act as the key environmental trigger for differentiation, and increases cAMP level and cAMP-mediated responses. For comprehensive understanding of cAMP signaling, we studied the enzymes related to cAMP metabolism. A stage-specific and developmentally regulated isoform of receptor adenylate cyclase (LdRACA) showed to regulate differentiation-coupled induction of cAMP. The soluble acidocalcisomal pyrophosphatase, Ldvsp1, was the major isoform regulating cAMP level in association with LdRACA. A differentially expressed soluble cytosolic cAMP phosphodiesterase (LdPDEA) might be related to infection establishment by shifting trypanothione pool utilization bias toward antioxidant defense. We identified and cloned a functional cAMP-binding effector molecule from L. donovani  that may modulate metacyclogenesis through induction of autophagy. This study reveals the significance of cAMP signaling in parasite survival and infectivity. Leishmania results in a spectrum of clinical manifestations referred to collectively as leishmaniases. The clinical manifestations range in severity from spontaneously healing cutaneous ulcers by L. major infection to potentially fatal visceral disease by L. donovani infection. The parasite is a digenic one and in its infective cycle, the parasite is transmitted as promastigote from the gut of insect vector female phlebotomine flies to mammalian hosts. The procyclic promastigotes get converted to metacyclic ones and are phagocytosed by mammalian macrophages where they convert into amastigote form, which is able to survive, and replicate within the phagolysosome. Along with a substantial alteration of nutrient availability, the parasite must adapt to new conditions of temperature and pH (37°C and pH 5.5) which acts as an initial environmental stress to the parasite. This physical conditioning has proved indispensable for Leishmania differentiation and in vitro transformation protocols are already in use mimicking the physical condition encountered in mammalian host [γ secreted by activated macrophages play important role in differentiation of naïve T-helper cells into proinflammatory Th1 subset. However, there are some intracellular parasites like Leishmania that are able to impair these activities by taking advantage of the host anti-inflammatory response to avoid self-damage by modulating its own biology and host environment to persist successfully inside the host. Even in the face of exposure to toxic prooxidants a subset of the Leishmania parasites that invades the host macrophages survives and subsequently converts into intracellular amastigotes, finally leading to disease manifestation [Infection by protozoan parasites of the genus ian host , 2. Afteian host . MacrophLeishmania, genes like superoxide dismutase, peroxidoxin, and trypanothione reductase are implicated in antioxidant defense against reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI) [Leishmania, in the absence of any known transcription factors, it is really unknown what triggers the expressions of the genes speculated to be associated with its antioxidant system. Interestingly, like in many lower organisms, environmental cues seemed to play some important roles in controlling biology of the parasite. Preexposure to environmental stress (pH 5.5 and temperature 37°C) has been shown to induce resistance against oxidative damage in this organism [Leishmania parasites to resist oxidative damage was observed to be coupled with their transformation to amastigote stage and there may be more than one mechanism of environmental sensing along with stress exposure, which finally trigger differentiation of the parasite. cAMP response has been implicated as one of the major environmental sensing machineries associated with stress response in many unicellular eukaryotes like Plasmodium, Trypanosoma, and others. cAMP in malarial parasite, Plasmodium falciparum triggers the conversion of asexual erythrocytic ring-stage parasites to sexual precursors, gametocytes [P. falciparum could synthesize its own cAMP by adenylate cyclase (AC) which is uniquely not stimulated by mammalian AC activator Forskolin or heteromeric G-protein activators AIF4. Moreover, cAMP signaling effector molecule Protein Kinase A (PKA) plays an important role in conductance of anions across the host cell membrane of Plasmodium-infected RBC [P. falciparum-infected RBC [Entamoeba histolytica triggers the phosphorylation of proteins involved in actin rear-arrangements necessary for adhesion and locomotion. Moreover, cAMP-response elements could play an important role in regulating actin expression and organization in signaling processes activated during tissue invasion. cAMP also plays an important role in Trypanosome differentiation from long slender form to short stumpy form, the form in which the cAMP level declines [2+, which seemed to have a receptor located in the membrane or as a part of AC. Also in Plasmodium, evidences identified intracellular Ca2+ store utilized by both melatonin and cAMP pathways. Also another component of cAMP signaling, the phosphodiesterases (PDEs) has transmembrane domains suggesting that they are integral membrane proteins. Hence, it was indeed necessary to study whether cAMP has similar importance in the parasite survival and infectivity. The Ras-cAMP pathway serves as a negative regulator of stress response in Saccharomyces cerevisae [ Few previous studies suggested that in es (RNI) –8. Disrues (RNI) –10. But,organism . The abietocytes . P. falccted RBC . It is ncted RBC . Activatdeclines . Moreoveerevisae , 17. Leishmania cAMP signaling and its control. We have tried to assess the functions of all the enzymes that are intimately associated with cAMP metabolism in the parasite  increases the resistivity of the parasites against induced oxidative stress by H2O2 and peroxynitrite [γ-activated macrophages with more efficiency than the parasites not exposed to differentiation condition [ynitrite . Moreoveondition . Since dondition . This inDictyostellium and Trypanosoma, more understanding was required regarding the role of cAMP in parasite survival in macrophage's hostile environment. Increasing intracellular cAMP level by cell permeable cAMP analog, pCPT-cAMP resulted in increased resistance against H2O2 and peroxynitrite. On the other hand, enhanced resistance by exposure to differentiation condition could be reversed by adenylate cyclase inhibitor, DDA (dideoxyadenosine), and PKA inhibitor, H89. To further ascertain the ability of cAMP in cytoprotection of the parasites against H2O2, three parameters were checked: DNA degradation, protein carbonylation, and ultrastructural analysis. The extent of DNA degradation and protein carbonylation by H2O2 was reduced in pCPT-cAMP-treated and differentiation condition-exposed cells, which got reversed by treatment with DDA, and H89 [Since cAMP plays a pivotal role in the differentiation, cell movement, and stress response in several organisms like  and H89 . SimilarLeishmania. Cell cycle was studied after intracellular cAMP concentration was modulated by treating the cells with pCPTcAMP, DDA and H89. pCPTcAMP caused a significant G1 phase arrest whereas treatment with DDA, and H89 decreased such arrested condition [Morphological transformation of promastigotes to amastigotes by exposure to 37°C and pH 5.5 occurs during cell cycle arrest at G1 phase . As resiondition . This daLeishmania has unique antioxidant machinery devoid of catalase and glutathione peroxidase. In most eukaryotic systems four enzymes have been implicated in antioxidant defense, namely, catalase, glutathione peroxidase, superoxide dismutase (SOD), and peroxidoxins (PXN). In Leishmania, instead of glutathione, trypanothione, a unique redox cycling glutathione-spermidine conjugate, is present, which in concert with trypanothione reductase (TR) maintains the intracellular reducing environment and resistance to reactive oxygen species (ROS). From genome analysis we know that at least 2 Sods and 3 different Pxns are present in Leishmania, of which Pxn1 is found to be differentially expressed and active against both ROS and reactive nitrogen intermediates (RNIs). A direct correlation between these antioxidant gene expression and intracellular cAMP response could be suggested from observations at both mRNA and protein levels of the genes, namely LdPxn1, LdSodA, and LdTr. They were all elevated by positive modulation of cAMP as well as on exposure to differential condition. Such upregulation of antioxidant genes of the parasite appears to be essential for induction of stress-resistance response of the parasite [Normally, cytoprotection in eukaryotes depends on a number of molecular machineries, the most important of which are antioxidant enzymes. parasite .Leishmania for the last 20 years. Reports suggested that there are more than 10 adenylyl cyclases in this parasite. This surprisingly high number of different adenylyl cyclases might be related to any other peculiarity of the parasite. Interestingly, there is no report of any G proteins in the parasite, and therefore, possible functions of adenylate cyclases are yet to be deciphered. Previously, two receptor adenylate cyclases from L. donovani (LdracA and LdracB) were analyzed which form part of a cluster of five similar genes. They were observed to be developmentally regulated with their expression in promastigote stage and not in amastigote stage [Only very few publications have addressed adenylate cyclases in te stage . Since cLeishmania lacks G-proteins, it was important to seek what provides a stringent control to the receptor adenylyl cyclases so that the strict local confinement of a cAMP signal, crucial for allowing local effect to occur, could be maintained. Many lower organisms were observed to have the total inorganic pyrophosphate pool (PPi) and polyphosphate pool (polyP) as environmental sensors. In Leishmania, control of adenylyl cyclases might be brought about by a further peculiarity of the parasite, that is, their high concentration of cytoplasmic PPi. This high concentration might effectively block cAMP synthesis via product inhibition of the adenylate cyclase reaction, the products being cAMP and PPi. Experiments were, therefore, designed to observe whether such PPi pool generates a negative feedback to receptor adenylyl cyclases towards formation of cAMP. The total PPi pool in log phase promastigote was found to be quite high (milimolar range), but it was interesting to note that differentiation condition exposure decreased the total PPi pool significantly by 1 hour. Modulation of PPi level might be largely due to the hydrolyzing enzyme, pyrophosphatase. Leishmania genome showed the existence of 3 different pyrophosphatases, namely, putative vacuolar type proton translocating pyrophosphatase (V-H+ ppase), soluble acidocalcisomal pyrophosphatase (LdVSP1), and putative inorganic pyrophosphatase (Ioppase). V-H+ ppase is known to be associated with acidocalcisomal membrane whereas vsp1 is soluble acidocalcisomal form and ioppase is of cytosolic localization. Expressions of all these pyrophosphatases were observed in both cytoplasmic and membrane fractions of L. donovani promastigotes by Western blot with antibodies raised against each of them by the administration of custom peptide in rabbit. V-H+ ppase was found to be predominantly present in membrane fraction of both normal and stress- (37°C and pH 5.5) exposed parasites. Its expression was not altered by the duration of stress exposure. Ldvsp1, on the other hand, was found to be present mainly in the cytoplasmic fraction of normal promastigotes. However, upon stress exposure, its expression was gradually enhanced in the membrane fraction with a maximum expression at 2 h after stress exposure with a plateau after 4 h. Further exposure did not alter LdVSP1 expression level . The putatively cytosolic inorganic pyrophosphatase (ioppase) was mainly detected in the cytoplasmic fraction with its expression unaltered after stress exposure. Expression pattern of LdVSP1 could provide some clue of its control on PPi pool.Since L. donovani promastigotes after stress exposure for various time periods by indirect immune fluorescence using antibodies raised against respective pyrophosphatases. As evidenced from immunofluoroscence, inorganic pyrophosphatase (ioppase) was cytosolic in both normal and stress-exposed cells. Neither did it colocalize with the acidocalcisome marker, nor it was found to be associated with any kind of membrane structure after stress exposure. The acidocalcisomal enzyme was found to be diminished significantly with time. LdVSP1 is primarily localized in vesicle-like structures of various sizes in normal unexposed promastigotes. Such cellular organization is typical of acidocalcisome distribution [+ ppase was found to be colocalized with acidocalcisomal marker in both normal and stressed conditions and V-H+ ppase being a membrane bound acidocalcisomal pyrophosphatase could not be visualized after 1 hour of stress. These observations point towards the fact that the soluble acidocalcisomal LdVSP1 moves towards membrane vicinity by change in acidocalcisome biogenesis and function during stress .Relative expression and localization of the different pyrophosphatase proteins were then assessed in ribution  and LdVSDuring stress, PPi pool was observed to get modulated, and interestingly, Ldvsp1 was found to be located near membrane vicinity after stress exposure, where leishmanial adenylyl cyclases (LdRACA and LdRACB) reside. To fully understand the regulation of leishmanial receptor adenylyl cyclases, experiment was carried to observe whether leishmanial adenylyl cyclases interact with LdVSP1 during stress. Observation indicated that LdVSP1 could interact with LdRACA but not with LdRACB during stress exposure . This indicated that one part of cAMP regulation might have been contributed by pyrophosphatase enzymes along with the total inorganic pyrophosphate pool modulating the function of leishmanial adenylyl cyclases. The high concentration of PPi might effectively block cAMP synthesis via product inhibition of adenylyl cyclase reaction, the products of which are cAMP and PPi. These observations agree well with the previous speculation that LdRAC enzymes might have their catalytic domains stuck in a soup of PPi, being so strongly downregulated that they need activation which our study showed to be the action of soluble acidocalcisomal pyrophosphatase LdVSP1 .Drosophila and Dictyostellium. Though, several class I isoforms have been identified in T. bruci and T. cruzi and PDE activity was previously reported in Leishmania, only very recently 2 PDEs have been cloned from L. major [L. donovani and inferred that LdPDEA is differentially expressed and decrease of cytosolic activity is due to PDEA downregulation. Kinetic analysis showed detectable reduction of PDE expression 6 hours after exposure to differentiation condition and this was supported by immunofluorescence analysis [μM for cAMP with no activity against cGMP. It was found to be a typical class 1 metal-dependent PDE . The mammalian PDE inhibitors could cause inhibition of this leishmanial PDE at very high concentrations barring 2 inhibitors (dipyridamole and trequinsin) proving it to be somewhat different from the mammalian counterpart [Since it could be speculated that intracellular cAMP pool is regulated by adenylate cyclase (AC) with the help of enzymes like pyrophosphatases, it was exigent to observe another part of cAMP regulation by phosphodiesterases (PDE) that hydrolyze cAMP to 5′-AMP or cGMP to 5′-GMP. Since PDE activity is contributed by several families of PDE of which some might be located in the immediate vicinity of LdRAC, we would discuss the concrete information availed by us studying the leishmanial phosphodiesterases. Depending on the catalytic properties, PDEs are classified into 3 different categories, namely, class I, class II, and class III. 21 genes for PDE have been identified in mammals and several in L. major . Since tL. major . We exteanalysis . We thennterpart . Leishmania is done by peroxidase as it lacks functional catalase. Since glutathione (GSH) is absent in Leishmania, peroxide action is mainly based on trypanothione (TSH), a glutathione-spermidine conjugate. Trypanothione is biosynthesized from arginine by arginase, ornithine decarboxylase (ODC), and other enzymes, which convert it to spermidine. It then conjugates with GSH. First we checked the availability of precursors like arginine and ornithine and found that the expression of arginine and ornithine transporter was not affected by PDEA inhibiton. Functional arginine and ornithine transport was also not affected [In order to look into the functional significance of LdPDEA we then silenced the gene using tetracycline-inducible knock-down system . When weaffected . But wheaffected . We, theaffected .2C2 heterotetramer consisting of two catalytic and two cAMP-binding regulatory subunits. Binding of cAMP to the regulatory subunits releases the active catalytic subunits, which are then free to phosphorylate a broad range of substrates. Recently, two PKA catalytic subunits (PKAC) from Leishmania have been cloned, characterized, and found to be sensitive to mammalian PKA inhibitors [Leishmania and perhaps plays a regulatory role in the parasite. In spite of the failure of previous attempts to identify PKA holoenzyme from kinetoplastidae parasites [T. cruzi [L. donovani [Leishmania as LdPKAR from L. donovani seemed to modulate metacyclogenesis, the process where the promastigotes get converted to infective form through induction of autophagy. Since regulatory subunits of PKA (PKAR) spatially and functionally interact with m-TOR during autophagosome maturation and deletion of PKAR results in activation of m-TOR leading to autophagic deficiency in mammalian cells and tissues [Though cAMP-dependent protein Kinase (PKA) is well characterized in eukaryote world, very little information is available on this particular downstream effector of cAMP signaling in the parasite. Our observations clearly indicated that temperature and pH stress which is responsible for transformation of promastigote to amastigote can also induce the PKA activity along with increasing cAMP levels. Moreover, substrate level phosphorylation on serine and threonine residues also increased during temperature and pH stress and in the case of positive modulation of cAMP by cell permeable cAMP analogs. PKA exists as inactive Rhibitors , 26 suggarasites , functioT. cruzi . To decidonovani . Moreove tissues , 31, ourE. coli BL21 (DE3) pLysS host. The fusion protein with an N- hexahistidine-tag was purified under nondenaturing conditions using Ni-NTA columns. It was found to be a single copy gene. The ORF of LdPKAR encodes a 502 amino acid polypeptide of molecular weight of 58.5 kDa. Comparison of protein sequences with Trypanosoma showed extensive identity with two of them, T. brucei (60%) and T. cruzi (66.1%), and showed 32.1% homology with bovine PKAR1-α. The N-terminal moiety of LdPKAR1 is longer than that of mammalian or S. cerevisiae PKARs and bears no identifiable functional domain. In analogy to other type 1 regulatory subunits, residues 133–137 and 203–207 probably represent the candidate pseudosubstrate sequences required for interaction and inhibition of PKA catalytic domain. Apart from this, residues 244–355 and 364–476 form the cyclic nucleotide binding domains A and B, respectively, which are composed of a number of conserved residues fitting the structural mode of bovine regulatory subunit PKAR1-α [Leishmania reportedly encodes two functional PKACs [The LdPKAR gene-encoding sequence was successfully cloned into the bacterial expression vector pET16b and expressed in al PKACs , and theγ-activated macrophages compared to wild-type parasites indicating greater infectivity of LdPKAR-over-expressing cells. LdPKAR appeared to have a role in the metacyclogenesis of L. donovani [LdPKAR expression was found to be increased in late stationary phase promastigotes, a condition metabolically similar to metacyclic promastigotes. In line with this observation, we found that the expression of LdPKAR significantly increases under starvation condition, a trigger for metacyclogenesis. Moreover, its overexpression could induce the onset of metacyclogenesis in the cells. Several properties like morphology, agglutination to PNA, increased expression of soluble acidocalcisomal pyrophosphatase (VSP1), sensitivity to human serum, and macrophage infectivity which distinguish metacyclic promastigotes from procyclic ones were all assessed to observe the role of LdPKAR on metacyclogenesis. Observations confirmed a definite role of LdPKAR on metacyclogenesis. Moreover, LdPKAR-over expressing cells were also found to be more efficient in surviving within IFN-donovani .S. cerevisiae [L. major encodes a single copy ATG8 gene (LmjATG8) and fluorescent tracking of ATG8 entails autophagy monitoring in Leishmania parasites as efficiently as in mammalian and yeast cells [L. donovani promastigotes. The significance of these observations with respect to cAMP signaling of the parasite for establishment of its infectivity seemed quite clear but further studies are required to decipher the intricate roles of all the components of leishmanial PKA [PKA activity serves as a regulator of autophagy in a number of mammalian cell lines and such regulation seems to be evolutionarily conserved as autophagy is negatively regulated by Ras/PKA pathway in revisiae . LdPKAR st cells . Percentst cells . Ultrastnial PKA .Leishmania parasites to macrophage oxidative damage is associated with increased intracellular cAMP and cAMP-mediated response. Parasites having increased cAMP-response elements were more cytoprotective, having higher levels of antioxidant enzymes and having more free radical scavenging capacity. For comprehensive understanding of cAMP signaling, we then studied the cAMP synthesizing enzyme, adenylate cyclase, the degrading enzyme phosphodiesterase (PDE), the regulatory enzyme pyrophosphatase (PPase) and the functional enzyme, cAMP-dependent protein kinase (PKA). Of 10 different leishmanial receptor adenylate cyclases (LdRACs), two (LdRACA and LdRACB) are stage-specific and developmentally regulated. Silencing and other biochemical parameters showed that differentiation-coupled induction of cAMP is regulated by LdRACA. We are the first to clone and characterize all five isoforms of PDE from Leishmania and showed that the soluble cytosolic isoform, PDEA, is heavily downregulated as the parasite is differentiated from promastigotes to amastigotes. Knockingdown the enzyme as well as by using specific inhibitors, we found that PDEA-inhibited parasites have markedly higher peroxide degradative capacity. This increased peroxide degradation is not due to increased trypanothione (TSH) biosynthesis or transport; rather it is due to the shifting of TSH pool utilization bias toward peroxide degradation, that is, antioxidant defense. Since pyrophosphate, one of the reaction product of adenylate cycase, is related with functionality of receptor adenylate cyclase, we studied the enzyme providing stringent control for it, that is, pyrophatases. Of the three different phosphatases present in Leishmania, the soluble acidocalcisomal form, LdVSP1 was found to be the major isoform regulating cAMP level and peroxide neutralizing capacity. The study on Ldvsp1 further suggests the significance of its association with LdRACA in regulating the cAMP pool which perhaps triggers the differentiation-associated events that ultimately affect the infectivity of the parasite  subunits have been cloned and characterized from different Leishmania species, but the regulatory subunit has not yet been characterized from any Leishmania species. We identified a regulatory subunit of PKA from L. donovani (LdPKAR), which is expressed in all life cycle stages. Its expression attained maximum level in stationary phase promastigotes which are biochemically similar to infective metacyclic promastigotes. Starvation condition, the trigger for metacyclogenesis in the parasite, elevates PKAR expression, and under starvation condition, promastigotes overexpressing PKAR attained metacyclic features earlier than normal cells. Furthermore, PKAR overexpression accelerates autophagy, a starvation-induced cytological event necessary for metacyclogenesis and amastigote formation (L. donovani that may modulate important cytological events affecting metacyclogenesis. Since no bona fide cAMP-binding protein of defined function has yet been identified in Leishmania or in any other kinetoplastidae, the biological significance and molecular mechanism behind cAMP signaling is still an open field to be explored.We first showed that differentiation-coupled induction of resistance of parasite . We thenormation . Conditi


Efficiently encoding sound features, e.g. temporal fine structure or envelope, is critical for perception tasks such as sound localization and identification. Whereas the auditory nerve (AN) fibers convey all information transduced at the cochlea, the pathway bifurcates in the cochlear nucleus, the first station after AN. The auditory system of the barn owl is a good example of such functional differentiation, where one subdivision of the cochlear nucleus  is thought to encode sound intensity information used for interaural intensity difference processing whereas the other (Nucleus Magnocellularis) encodes temporal fine-structure information necessary for interaural time difference processing .The fact that NA responses to frozen broadband sounds are very reproducible on a slow time-scale  led to tBecause the AN encodes sounds through frequency-selective channels that exhibit low-pass modulation sensitivity the question then arises as to how NA cells, receiving a few around 5 ) AN inpu  AN inpu


Antimicrobial peptides constitute a diverse class of naturally occurring antimicrobial molecules which have activity against a wide range of pathogenic microorganisms. Antimicrobial peptides are exciting leads in the development of novel biocidal agents at a time when classical antibiotics are under intense pressure from emerging resistance, and the global industry in antibiotic research and development stagnates. This review will examine the potential of antimicrobial peptides, both natural and synthetic, as novel biocidal agents in the battle against multi-drug resistant pathogen infections. Staphylococcus aureus infections have risen dramatically in recent years [Staphylococcus aureus infections classified as methicillin resistant and 30% of enterococci exhibiting vancomycin resistance [Despite continuing efforts, the increasing prevalence of resistance among pathogenic bacteria to common antibiotics has become one of the most significant concerns in modern medicine. With significantly reduced investment in antimicrobials research and development among major pharmaceutical companies, novel alternatives to existing treatment strategies are not being produced at a sufficient rate to keep pace with the emergence of resistance and the supply pipeline runs perilously close to drying up . Incidennt years , with alsistance . In addisistance .One area of antimicrobial drug research that does shows significant promise is in the discovery and development of antimicrobial peptides (AMPs). Antimicrobial peptides in nature serve as important defensive weapons throughout the animal and plant kingdoms against a broad spectrum of bacterial and fungal pathogens . SourcesAntimicrobial peptides are short , exhibit rapid and efficient antimicrobial toxicity against a range of pathogens ,15 and cStaphylococcus aureus. Lipoglycopeptide derivatives of vancomycin in development include oritavancin and dalbavancin which possess increased activity against vancomycin-resistant strains [The majority of antimicrobial peptides are cationic with more than a thousand characterized and are thus termed cationic antimicrobial peptides (CAPs) . Natural strains . Slight  strains . Cationi strains .Although the secondary structures that antimicrobial peptides adopt may vary between classes they share the same characteristic of developing an amphipathic structure and being cationic under physiological conditions ,31. SecoXenopus laevis [These peptides are of particular interest as one face of the helix structure contains a majority of hydrophobic residues whereas the opposite face contains mainly polar amino acids allowing efficient solubilization of microbial membranes . Amphipas laevis  and the s laevis . Cyclic s laevis .Extended structures with a high regularity of one or two amino acid residues, such as proline, glycine, histidine or tryptophan, make up the remainder of the four major structural varieties ,39. ExteDespite the vast majority of antimicrobial peptides being cationic in nature, a significant number of anionic AMPs have been reported; serving as important weapons in the eukaryotic innate immune response –48. Pept2+), as cofactors for biocidal activity [2+ [A disadvantage of many anionic antimicrobial peptides is that they often require cations, for example zinc . In frogs, variations in the sequence and spectrum of activity of these peptides are considerable with hundreds of characterized peptides providing protection against a vast range of bacterial and fungal species [Bombina [Xenopus [Rana [Phyllomedusa [Litoria [Bombina maxima, commonly known as the Chinese red belly toad has as many as forty genes linked to the production of different antimicrobial peptides [et al. showed that Bombina maxima produced a group of peptides called maximins that demonstrated minimum inhibitory concentration (MIC) values in the μg/mL range against a broad spectrum of microbial and fungal pathogens including Staphylococcus aureus, Escherichia coli, Bacillus dysenteriae, Klebsiella pneumoniae and Candida albicans [Staphylococcus aureus. Maximin-4 consists of twenty seven amino acids and has the potential to be produced synthetically via SPPS. Dermaseptins are a family of linear lysine-rich cationic antimicrobial peptides derived from the genus Phyllomedusa. They consist of between twenty eight and thirty four amino acid residues and have been shown to inhibit the growth of a wide variety of Gram-positive and Gram-negative bacteria, fungi, protozoa and yeast [Structural characterization has facilitated their synthetic production  species ,68,69. T species ,70. Rele[Bombina , Xenopus[Xenopus , Rana [7us [Rana , Phyllomlomedusa  and Lito[Litoria . The spepeptides . Researcalbicans . Both mand yeast ,77.The diverse range of antimicrobial peptides available in nature provides a vast scope for the design of novel and improved synthetic variations. Structure and its effect on the hydrophobic:charge ratio is more important with regard to antimicrobial activity than size and the manipulation of primary amino acid sequence can improve factors such as specificity, toxicity and stability ,78. Ultret al. have proven that tryptophan provides better bulk and lipophilicity than tyrosine and a minimum of two bulk and two charged residues are required to be present to give activity against staphylococci, with Escherichia coli requiring an additional bulk tryptophan. Arginine also acts a better source of charge than lysine within the minimum motif [et al. reported however that peptides containing four amino acids would find it difficult to form a characterized, stable amphipathic structure, therefore questions still relate to their mode of action. Reported results also show that the balance of hydrophobicity and charge play an important role just as in the larger antimicrobial peptides. By altering both, the amino acid residues and the acyl chain length, it was found that increasing hydrophobicity and/or charge is not necessarily indicative to increasing antimicrobial function [Strom um motif . Howeverum motif ,81. The um motif . Small aum motif . Previoufunction .in vivo, with many peptides limited to use as topical applications. Human chymotrypsin-like enzymes function at basic amino acid residues [d-enantiomers thus a more viable alternative is to select unnatural amino acids, such as ornithine. The use of ornithine as a charged moiety is preferable as the use of a non-coded amino acid provides stability against proteases [et al. They demonstrated the potent action of ultrashort tetrapeptides with two ornithines representing the charged portion and two tryptophans providing bulk and hydrophobicity [α-amino terminus of ornithine. The incorporation of ornithine into the lipopeptide scaffold has also been tested in the synthetic peptide MSI-843 [Staphylococcus aureus; Gram-negative Pseudomonas aeruginosa and Escherichia coli and the fungus Candida albicans.Host and microbial proteases and the unfavorable pharmacokinetics they bring present a major barrier to the use of peptides as antimicrobials residues , thereforesidues . As protresidues . L-stereresidues . Antimicroteases . Examplehobicity . Modific MSI-843 . ContainStaphylococcus aureus resistant to common antibiotic regimens [et al. produced a series of smaller dipeptides of a general formula XRY where; X represented a variant of a bulky amino acid with a lipophilic side chain, for example cyclohexylalanine; R represented arginine, the charged moiety; Y represented a C-terminal ester or a lipophilic amide derivative [tert-butyl-tryptophan-arginine-benzyl amide was shown to be the most active peptide in the study. This structure correlates with the minimum requirement for two units of bulk, provided by the tryptophan derivative and benzene ring of the benzyl amide and two units of charge imparted by arginine’s guanidino group and the free Nα-amino terminus. Central to this theory Bisht et al. produced a series of ornithine and tryptophan containing tetrapeptides and evaluated them against planktonic forms of various Gram-positive and Gram-negative bacteria [Ultrashort cationic antimicrobial peptides consist of approximately four or five amino acids residues, with amino acid selection fulfilling the minimum range of properties required for effective antimicrobial activity to take place. The use of ultrashort cationic antimicrobial peptides is advantageous in terms of manufacture with reduced production costs and synthesis times. Their production correlates to a trend amongst peptide companies that has seen an increase in the production of these smaller peptides in relation to larger, more expensive natural peptides ,91. Haugregimens ,92–97. Oregimens ,78. Naturegimens . Tetra aregimens . With thrivative . Both X bacteria . To incri.e., fatty acid) with inherent antimicrobial activities represents an approach to the development potent antimicrobial agents, whereby spectrum of activity may be modulated via modification of the N-terminal substituent, whilst circumventing the commercial barriers associated with the manufacture of natural antimicrobial peptides in high yields.Lipopeptides are essentially a peptide attached to a lipid moiety. Such a peptide may include an antimicrobial peptide. Native lipopeptides are formed in bacteria and fungi non-ribosomally by cultivation on a range of carbon sources to form complex cyclic structures . They noA variety of anionic and cationic lipopeptides are currently licensed for commercial use, thus highlighting the clinical potential of these antimicrobials. These include daptomycin, polymyxin B and polymyxin E (colistin).Streptomyces roseosporus (Actinobacteria). Daptomycin is a cyclic depsipeptide consisting of thirteen amino acids, which includes three D-amino acid residues  linked to a hydrocarbon tail ten carbons in length derived from decanoic acid  involved in Undecaprenyl phosphate-4-amino-4-deoxy-L-arabinose production are controlled by the two component quorum sensing systems PhoP/PhoQ and PmrA/PmrB; and/or the RcsA/RcsB/RcsC system as outlined in Extracellular DNA may also play a role in increased resistance of biofilm forms of rabinose . Resistain vitro results and animal studies [in vivo and the large scale production costs of these peptides. It is expected that future research will allow the area of antimicrobial peptides to be harnessed therapeutically with the same degree of evolutionary success as they are utilized in nature as components of innate immunity. Antimicrobial peptides fulfill a number of the criteria expected from an ‘ideal’ biocide, namely performance , environmental fate (facile bioremediation), safety and cost.As biocides antimicrobial peptides have the potential to eradicate the most resistant forms of clinically relevant biofilm forming pathogens. Lipopeptides such as polymyxin B and daptomycin are already utilized in topical formulations , therefo studies ,223,224.


Vaccinating children aged 6–23 months, plus all other children at high-risk, will likely be more effective than vaccinating all children against influenza. We estimated cost-effectiveness of annually vaccinating children not at high risk with inactivated influenza vaccine (IIV) to range from US $12,000 per quality-adjusted life year (QALY) saved for children ages 6–23 months to $119,000 per QALY saved for children ages 12–17 years. For children at high risk  ages 6–35 months, vaccination with IIV was cost saving. For children at high risk ages 3–17 years, vaccination cost $1,000–$10,000 per QALY. Among children not at high risk ages 5–17 years, live, attenuated influenza vaccine had a similar cost-effectiveness as IIV. Risk status was more important than age in determining the economic effects of annual vaccination, and vaccination was less cost-effective as the child's age increased. Thus, routine vaccination of all children is likely less cost-effective than vaccination of all children ages 6–23 months plus all other children at high risk. During the 2003–04 influenza season, reports of >150 influenza-associated deaths among children and unprecedented demand for influenza vaccine highlighted the need to reevaluate the nation's influenza vaccination policies regarding children  influenza vaccine (LAIV) for healthy persons ages 5–49 years, which has a higher list price than the inactivated (injected) vaccine but is also potentially more effective .Using standard software , we created a mathematical model (decision tree) to estimate the effect of influenza vaccination on outcomes and costs among children. The decision tree evaluated 3 options: 1) no vaccination; 2) inactivated influenza vaccine (IIV); and 3) live, attenuated influenza vaccine (LAIV). It estimated costs and outcomes for influenza-related illness for children stratified into 10 subgroups by age  and risk status (high risk or not at high risk). Children were defined as being at high risk for influenza-related complications due to preexisting medical conditions .Influenza-related outcomes included in the decision tree were episodes of influenza illness , otitis media, mild pneumonia (and other complications treated on an outpatient basis), hospitalizations (with and without long term sequelae), and deaths. Event rates, by age and risk group, were derived from the published literature and were supplemented by expert opinion where data were limited or unavailable  (11–15) ,Inactivated vaccine was considered for all 10 subgroups, and LAIV was considered only for children not at high risk. Children 6 months to 4 years were included as a theoretical intended population for LAIV, although LAIV is currently licensed in the United States only for children 5–17 years. The most likely estimate for vaccine effectiveness against symptomatic influenza illness was lower for IIV (0.690) than the most likely estimate for LAIV (0.838)  .Adverse events attributable to influenza vaccination included in the decision tree were medically-attended episodes of injection site reactions, systemic reactions (defined as fever within 2–7 days of vaccination), anaphylaxis, and Guillain-Barré syndrome . Probabihttp://www.bls.gov/cpi/). Costs of physician visits for influenza illness, influenza-related hospitalizations, and vaccination-related adverse events were calculated by using a large database that reported payments for health insurance companies in the mid-Atlantic states of the United States . Vaccination costs included vaccine dose costs, administration costs, medical attention for vaccine adverse events, and, if an additional visit was required, parent time costs  and opportunity costs (parent time costs) for physician visits . All cosIt is recommended that first-time recipients aged 6 months through 8 years receive 2 doses of influenza vaccine  (20).,,The model projected 4 different outcomes that were averted through vaccination: influenza episodes, hospitalizations, deaths, and QALYs. The QALY is a measure of net health effects that takes into account the health benefits of averted influenza cases as well as the health costs of vaccination-related adverse events. We obtained QALY valuations for each health event in the model from 2 studies  .The primary outcome measure was the incremental cost-effectiveness ratio in dollars per QALY. Secondary measures included costs and events averted per 1,000 vaccinated children, dollars per influenza-related event avoided, dollars per hospitalization avoided, and dollars per death averted. One-way sensitivity analyses were conducted on all variables, in which the impact on the average $/QALY saved was examined by altering each variable within the range of given values . Two-wayInfluenza vaccination with IIV was projected to be cost saving for children ages 6–35 months at high risk and to require a net investment for all other age and risk groups. The projected benefits of vaccination decreased as age increased . For exaAll vaccination strategies had net positive QALYs gained, which indicated that the health benefits of vaccination outweighed the risks . For chiIIV use was cost saving among children at high risk ages 6 months to 2 years . For chiUsing base-case vaccine purchase prices for LAIV and IIV , LAIV foProbabilistic sensitivity analysis provided confidence intervals for projected costs and events averted and quasi-confidence intervals for cost-effectiveness ratios. By using base case assumptions, results for LAIV are slightly more favorable than IIV (compared to no vaccination), and vaccination with LAIV is the preferred strategy. However, probabilistic sensitivity analysis indicated projected results were similar for IIV and LAIV.Cost-effectiveness acceptability curves generated through probabilistic sensitivity analysis are very similar for IIV and LAIV C. The prIn 1-way sensitivity analyses, cost-effectiveness ratios were most sensitive to changes in influenza illness attack rate, hospitalization rates, total vaccination costs, and vaccine effectiveness . Cost-efTwo-way sensitivity analysis on influenza illness rate and vaccine effectiveness (IIV) resulted in changes in the cost-effectiveness ratio from a decrease in 11% for a season with a high influenza illness rate (35%) and high vaccine effectiveness (IIV) to an increase of more than a factor of 30 for seasons with a low influenza illness rate and low vaccine effectiveness (IIV). Combining a high influenza illness rate (35%) with low vaccine effectiveness (IIV) resulted in cost-effectiveness ratio ≈3 times base case results  for children not at high risk ages 6–23 months. Two-way sensitivity analyses on influenza illness rate and total vaccination costs (IIV) had similar results, ranging from a decrease in 6% in the cost-effectiveness ratio to an increase 25 times as high as the base case for seasons with low influenza illness rate and high vaccination costs (IIV). Two-way sensitivity analyses for vaccination costs and effectiveness yielded a 20% lower cost-effectiveness ratio for low costs and high effectiveness of vaccination (IIV) to 7 times the base case for high costs and low vaccine effectiveness (IIV).,–We found that influenza vaccination of children, both those at high risk and those not at high risk, in all age groups would have health benefits that outweigh vaccine adverse events as measured by QALYs for both IIV and LAIV. For children not at high risk ages 6 months–4 years, we estimated that influenza vaccination with IIV would cost <$25,000 per QALY saved. In comparison, other routinely-used preventive interventions, such as pneumococcal conjugate vaccination, cost an average of $7,000/QALY for children <2 years  and to vaccine effectiveness indicates that the cost-effectiveness of influenza vaccination can vary considerably from year to year. In seasons with a low influenza attack rate, the cost-effectiveness of vaccination with IIV would be dramatically higher than in the base case . The 2-wOur study contributes valuable new information because it incorporates survey-based health state preferences for influenza-related illness and vaccine adverse events. These preferences, which are expressed as QALYs saved, are important for 2 reasons. First, we were able to evaluate the net health benefits of vaccination by subtracting the QALYs lost due to vaccine adverse events from the QALYs gained due to averted influenza cases. The results suggest that vaccination of all children is desirable from a health standpoint. Second, the outcome measure of dollars per QALY saved allows policymakers to compare the cost-effectiveness of influenza vaccination of children with other potential investments in preventive health services.,,,,,–Authors of other economic analyses of influenza vaccination in children concluded that vaccination was more cost-effective than we found in our study (In a recent study that used cost-benefit analysis to evaluate the economics of influenza vaccination in children, Meltzer et al. arrived at conclusions similar to this analysis for many of the age/risk groups under consideration (Some studies that used mathematical models have suggested substantial community herd immunity effects from vaccinating school-aged children (,A recent randomized trial suggests that influenza vaccination has little, if any, effect on otitis media in children (The time-tradeoff questions we used to elicit preferences for health states differ from that commonly used for adult illnesses because the loss of quality of life for both parent and child are explicitly included. In addition, parents were asked to include the value of productivity losses to paid or unpaid work for caring for a child with influenza in the time-tradeoff amount; therefore, productivity losses were included in the health state quality adjustments, whereas parent time costs for vaccination were included as dollar costs. As a result, the time-tradeoff amounts presented here are not directly comparable to utility values from generic utility instruments for measuring reductions in quality-of-life for chronic health states, such as the Health Utilities Index (Recent data show that some influenza-related deaths in children may occur outside the medical setting (,Few data are available to guide assumptions on what proportion of children who experience mild systemic symptoms after vaccination, such as fever or respiratory symptoms, will see a physician. In the absence of reliable data, we selected an assumption that would be more likely to bias against vaccination rather than for and assumed it would be the same as the proportion of children who would visit a physician due to influenza illness. If the number of medically attended, vaccination-related adverse events were lower, the cost-effectiveness ratios would also be slightly lower, but cost-effectiveness results are not very sensitive to this parameter. We did not include any quality adjustment for vaccination itself aside from negative effects of vaccination-related adverse events. If vaccination itself were associated with a decrease in quality of life, cost-effectiveness ratios would be less favorable than in the current analysis. Previous analyses of other vaccinations, which included a quality adjustment for fever and fussiness following vaccination, were not sensitive to this parameter (Routine annual influenza vaccination using IIV for children age >2 years not at high risk is likely to result in net health benefits, but cost-effectiveness ratios are likely to be less favorable than for children ages 6–23 months and children of any age with a high-risk condition. Cost-effectiveness among children decreases with increasing age, although risk status is more important than age in determining the economic impact of annual influenza vaccination. Further work is needed to assess the potential impact of herd immunity on the cost-effectiveness of expanding influenza vaccine recommendations.


In vivo studies have correlated changes in mechanical load to modulation of the extracellular matrix and have indicated that increased mechanical force contributes to the enhanced expression and deposition of extracellular matrix components or fibrosis. Pathological fibrosis contributes to dysfunction of many organ systems. A variety of in vitro models have been utilized to evaluate the effects of mechanical force on extracellular matrix-producing cells. In general, application of mechanical stretch, fluid flow, and compression results in increased expression of extracellular matrix components. More recent studies have indicated that tissue rigidity also provides profibrotic signals to cells. The mechanisms whereby cells detect mechanical signals and transduce them into biochemical responses have received considerable attention. Cell surface receptors for extracellular matrix components and intracellular signaling pathways are instrumental in the mechanotransduction process. Understanding how mechanical signals are transmitted from the microenvironment will identify novel therapeutic targets for fibrosis and other pathological conditions.The biomechanical environment plays a fundamental role in embryonic development, tissue maintenance, and pathogenesis. Mechanical forces play particularly important roles in the regulation of connective tissues including not only bone and cartilage but also the interstitial tissues of most organs.  Mechanical forces play integral roles in embryonic development, homeostasis, and pathogenesis. All cells in multicellular organisms are exposed to mechanical forces of varying degrees. Endothelial cells, for instance, are exposed to shear stress due to the passage of fluid through the cardiovascular system. Chondrocytes and other cells in joints are exposed to repetitive compressive forces. The effects of mechanical forces on cells and tissues have received greater attention as models have been developed to systematically analyze these effects. Many of the early studies in this regard were focused on cells and tissues that are influenced by obvious mechanical force including the cardiovascular and musculoskeletal systems. Early investigations in the mechanobiology field relied on relatively simple and imprecise systems. For instance, studies have utilized a hanging-drop culture system to examine the effects of tensile forces on connective tissue cells . As intein vitro model systems were developed to more precisely isolate the effects of mechanical forces on cellular processes. Various systems were engineered to apply uniaxial or multiaxial distension or stretch to cells grown on deformable substrata. These systems date back several decades to studies conducted on smooth muscle cells that were cultured on deformable elastin matrices [The mechanobiology field began to move forward rapidly as matrices , 5. Amonmatrices –9. It haStudies by Leung et al.  were amoin vitro testing systems the effects of these forces and the mechanisms of their actions have been and continue to be investigated.The expression of ECM components is regulated by diverse biochemical factors including growth factors, cytokines, and hormones , compression, and shear stress. A number of early studies utilized cells cultured on deformable membranes to examine the cellular effects of mechanical stretch. These studies illustrated that mechanical stretch of isolated cells mimicked many of the responses that had been characterized to increased load proteins . in vivo had been known for some time to impact synthesis and deposition of the ECM. For instance, increased cardiovascular load has for some time been correlated to increased deposition of ECM components. The period immediately after birth is associated with increased cardiovascular load and rapid growth of the heart [in vivo, all have generally illustrated that mechanical stretch of matrix-producing cells (largely fibroblasts and smooth muscle cells) results in increased production of ECM components or a profibrotic response [Alterations in mechanical load he heart . This pehe heart –18. Incrhe heart , 20. Whiresponse –24. in vivo environment, apparatuses are being developed to investigate mechanical forces in three-dimensional in vitro systems. Several recent studies have applied mechanical loads to cells cultured in three-dimensional scaffolds [To more accurately mimic the caffolds , 26. Thein vitro systems have been invaluable in elucidating the effects of mechanical forces on cells and the mechanisms of mechanotransduction, cells function within a three-dimensional environment whose mechanical properties can change during development [While two-dimensional elopment  or varioelopment , 29, canelopment –34, and elopment . Due to elopment . Accumulelopment . Componeelopment , shape [elopment , migratielopment , 41, andelopment , 43.α1(I), α2(I), and α1(III) collagen mRNAs were diminished in cells cultured in the three-dimensional scaffolds. Further studies by this group of investigators illustrated that collagenase activity is enhanced by culture in three-dimensional scaffolds promoting a collagenolytic phenotype in the less rigid environment of the collagen gels [α1(I) and α1(III) mRNA expression paralleled proliferation with increasing expression in stiffer regions of the scaffold. The expression of prostaglandin, which is an endogenous antifibrotic factor, was opposite to that of the collagens with increased levels in the less rigid portions of the construct. These studies and others indicate that the biomechanical properties of the microenvironment can direct the expression of ECM components and ECM-modifying enzymes with stiffer tissue properties contributing to enhanced ECM production. Less rigid matrices appear to promote an anti-fibrotic environment that includes increased production of matrix-degrading proteases and anti-fibrotic agents like prostaglandin.Seminal studies by Mauch et al.  were amogen gels . A numbegen gels . Similargen gels . Liu et gen gels  have uti2+ on softer substrates stimulated fibronectin assembly illustrating that integrin activity is an important mediator of this process (discussed further below). Previous studies have illustrated that the conformation of fibronectin on more rigid substrata is extended, which exposes additional binding sites for cells to fibronectin [Matrix rigidity impacts not only the expression of ECM components but also other parameters associated with fibrosis including the deposition and organization of these components. Studies by Halliday and Tomasek  illustraronectin . This isronectin . α-smooth muscle actin. Myofibroblasts are responsible for alterations to connective tissues including increased synthesis of ECM components. In addition, these cells produce cytokines and growth factors that promote the fibrotic response in an autocrine/paracrine manner. Myofibroblasts are derived from a variety of cells in response to tissue damage and stress including quiescent fibroblasts, blood-derived fibrocytes, mesenchymal stem cells, stellate cells of the liver, and others [An important step in tissue fibrosis of many organs is the formation of myofibroblasts or myofibroblast-like cells. These cells are characterized by enhanced contractile activity, formation of stress fibers, and expression of d others , 53. Regd others . Stiffenα-smooth muscle actin expression, routinely used as a marker for myofibroblast formation. Culture of cells in free-floating collagen gels  yielded the lowest relative level of α-smooth muscle actin expression. Similar results have been obtained in experiments examining matrix rigidity and differentiation of bronchial fibroblasts to a myofibroblast phenotype [α-smooth muscle actin and interstitial collagen expression. In the former studies, the expression of the α1 and α2 integrins, which are collagen receptors, correlated to enhanced myofibroblast formation on collagen-coated plastic [α-smooth muscle actin expression between cells in free-floating and constrained collagen gels cultured in low serum (5%); however, enhanced α-smooth muscle actin expression was seen in constrained gels at higher serum levels (10%). These studies and others illustrate integration of mechanical and biochemical signals by cells.Studies using a three-dimensional collagen scaffold system illustrated that collagen deformability or compliance is inversely related to the transformation of cells into a myofibroblast phenotype . Culturehenotype . Culturehenotype . Increas plastic . Incubat plastic . In thesβ to the culture medium enhanced differentiation on stiff scaffolds, again indicating interactions between the mechanical and biochemical environments. These studies also illustrated that interactions between the cells and the surrounding ECM and generation of mechanical tension are critical to the conversion to a myofibroblast phenotype. That is, coating of polyacrylamide scaffolds with collagen or fibronectin promoted myofibroblast formation to a much greater degree than polyacrylamide scaffolds coated with poly-L-lysine. Cell adhesion to poly-L-lysine is through electrostatic charges and not via specific integrin receptors. Studies with foreskin fibroblasts have illustrated that alterations in integrin expression accompany changes in substrate rigidity and myofibroblast formation [α-smooth muscle actin and primarily the α2β1 integrin. Culture of cells on more rigid substrata resulted in enhanced expression of α-smooth muscle actin and a switch to expression primarily of αvβ3 integrin.The conversion of hepatic stellate cells to a myofibroblast phenotype is a critical step in liver fibrosis and is part of the pathway to cirrhosis in chronic liver disease. Culture of hepatic stellate cells on tissue culture plastic and in high levels of serum results in their spontaneous conversion to a myofibroblast phenotype . Cultureormation . In thesin vitro [Fibroblasts isolated from diseased patients or animal models typically retain characteristics of their altered phenotype in vitro . Indeed,in vitro . Howeverin vitro . Cultureα-smooth muscle actin-containing cells. Interestingly, and of potential therapeutic significance, the proportion of myofibroblasts in the scaffolds decreased by approximately half when the elastic modulus was decreased by photodegradation. This coincided with a reduction in connective tissue growth factor and in proliferation. The classic dogma has been that once fibrosis has begun, it cannot be reversed; however, recent studies have illustrated that fibrosis can be halted or even reversed depending upon the extent of its progression [Recent studies have been carried out utilizing a novel photodegradable cross-linker-polyethylene glycol scaffold in which exposure to ultraviolet light can modulate the mechanical properties of the substratum to evaluate the effects on myofibroblast conversion of heart valve interstitial cells . Similargression . The aboμm [α-smooth muscle actin compared to adult fibroblasts. Inclusion of negatively charged, polyelectrolyte-coated gold nanorods within three-dimensional collagen gels significantly reduced the ability of neonatal cardiac fibroblasts to contract these gels and was accompanied by a significant decrease in both the expression of α-smooth muscle actin and type I collagen [in vitro assembly to type I collagen, and rheological characterization of the mechanical properties of these constructs revealed that these gels were stiffer and more elastic than controls or gels containing positively charged gold nanorods [Recent studies with gold nanoparticles have shown that they can be used for both measuring cell-induced deformation of the ECM as well as modulating matrix stiffness and formation of myofibroblasts. Stone et al.  describeμm . Recentlμm . While ncollagen . This stnanorods . These lAnother mechanism to take advantage of matrix mechanical properties therapeutically is in targeting death of cells via alterations in matrix rigidity. It has long been known that interactions with the ECM are necessary for survival of normal cells. However, the effects of the mechanical properties of the ECM on cell survival are only recently being addressed. Using polyacrylamide gels of varying rigidity coated with type I collagen, Wang et al.  illustraThe ability of matrix mechanical properties to direct cell behavior is also being integrated into novel tissue engineering approaches, particularly in attempting to develop vascularized tissue constructs . Examinain vitro systems have provided fundamental information regarding the molecular mechanisms whereby cells detect and respond to mechanical forces. During the past two decades, extensive progress has been made in understanding “mechanotransduction” or the mechanisms whereby physical stimuli are converted into chemical signals by cells [α/β heterodimer combinations, and specific α/β heterodimers serve as receptors for particular ECM ligand(s). The response of cells to mechanical stretch varies depending upon the ECM substratum suggesting a role for specific integrin heterodimers [α4 and α5 chains) or arginine-glycine-aspartic acid (RGD) peptides to prevent integrin-ECM interactions, MacKenna et al. [Studies utilizing by cells , 78. Desby cells . Integriby cells –82. Therrodimers , 83. Utia et al.  were amoα-actinin [These early studies set the stage for extensive research focused on the mechanisms whereby cells detect mechanical changes in the microenvironment and transduce these into biochemical and molecular alterations in the cytoplasm and nucleus. The cell-ECM linkage involving integrins and a myriad of associated proteins is a critical component of this process . It has -actinin , 90.A number of signaling molecules associate directly or indirectly with the integrin cytoplasmic domain including focal adhesion kinase (FAK). FAK was initially identified as a Src kinase substrate , 92. As Exposure of cells to mechanical force results in activation of numerous intracellular signaling pathways including protein kinases such as protein kinase C, c-Jun N-terminal kinases (JNK), extracellular signal-regulated kinases (Erk), and others  and transcriptional coactivator with PDZ-binding motif (TAZ) , 102. Anturnover . Several recent studies have begun to evaluate the functional roles of YAP and TAZ in mediating the response of cells to mechanical forces. In humans, the trabecular meshwork of the eye is approximately twentyfold stiffer in individuals with glaucoma than in normal individuals . Cells fin vivo three-dimensional environment. This has led to development of three-dimensional models to evaluate the effects of mechanical forces in a more in vivo-like environment. The realization that the biomechanical properties of the microenvironment can promote fibrosis and other responses has led to renewed interest in the effects of mechanical forces on cell and tissue behavior.It has become increasingly clear that most cells in the vertebrate body are exposed to varying degrees of mechanical forces. These forces impact embryonic development, homeostasis, and pathological conditions including fibrosis. Historically most of the studies that focused on mechanical force as a profibrotic stimulus utilized two-dimensional stretch or compression models with isolated matrix-producing cells. These studies have provided substantial knowledge regarding the responses of cells to mechanical force and the underlying mechanisms of this response. However, these systems do not adequately mimic the While extensive knowledge has been gained regarding the effects of the mechanical environment on cells and tissues, many questions remain regarding the molecular mechanisms of these effects. Identification of novel mechanoresponsive proteins such as YAP and TAZ will provide new therapeutic targets to modulate the deleterious effects of increased mechanical force. As it is becomingly increasing clear that tissue stiffness may precede fibrosis or at least contribute to ongoing fibrosis, identifying methods to modulate the mechanical properties of the microenvironment may also yield novel therapeutic approaches. Along these lines, specific nanomaterials may provide such reagents. However, the mechanisms whereby these materials regulate tissue properties have not been elucidated.


Metabolite profiles can be used for identifying molecular signatures and mechanisms underlying diseases since they reflect the outcome of complex upstream genomic, transcriptomic, proteomic and environmental events. The scarcity of publicly accessible large scale metabolome datasets related to human disease has been a major obstacle for assessing the potential of metabolites as biomarkers as well as understanding the molecular events underlying disease-related metabolic changes. The availability of metabolite and gene expression profiles for the NCI-60 cell lines offers the possibility of identifying significant metabolome and transcriptome features and discovering unique molecular processes related to different cancer types.We utilized a combination of analytical methods in the R statistical package to evaluate metabolic features associated with cancer cell lines from different tissue origins, identify metabolite-gene correlations and detect outliers cell lines based on metabolome and transcriptome data. Statistical analysis results are integrated with metabolic pathway annotations as well as COSMIC and Tumorscape databases to explore associated molecular mechanisms.Our analysis reveals that although the NCI-60 metabolome dataset is quite noisy comparing with microarray-based transcriptome data, it does contain tissue origin specific signatures. We also identified biologically meaningful gene-metabolite associations. Most remarkably, several abnormal gene-metabolite relationships identified by our approach can be directly linked to known gene mutations and copy number variations in the corresponding cell lines.Our results suggest that integrative metabolome and transcriptome analysis is a powerful method for understanding molecular machinery underlying various pathophysiological processes. We expect the availability of large scale metabolome data in the coming years will significantly promote the discovery of novel biomarkers, which will in turn improve the understanding of molecular mechanism underlying diseases. Metabolites are end products of cellular processes. Thus the profile of metabolites provides a snapshot of the physiological state of a cell complementary to its transcriptome and proteome, which manifests the functional status of events which occur more upstream. Since the size of metabolome is about 1-2 orders of magnitude smaller than transcriptome and proteome, the steady state concentration of a metabolite usually reflects the combined effects of multiple upstream factors. Conceivably, this property makes metabolites better biomarkers in some situations. In addition, identifying interrelations between metabolites and genes, proteins, or genome structures can potentially facilitate the elucidation of molecular mechanisms involved in pathophysiological processes.Previous work has revealed significant associations between gene and metabolite expression profiles, which added another layer of quantitative inferences to gene-wise correlations -3. Nam eConceivably, cancer samples provide excellent opportunities for identifying metabolic biomarkers and gene-metabome relationships due to the dramatic function alterations at the molecular level in cancer tissues. For example, cancer tissues usually exhibit more than 10-fold changes in the expression level of many genes in numerous microarray studies. Recently, the collaborative NCI60 project from the Developmental Therapeutics Program (DTP) of the National Cancer Institute (NCI) has made extensive measurements of various ‘omics’ data publicly available, including microarray, metabolomics, proteomics, etc. While the number of metabolome of metabolome-related studies and biomarker discoveries associated with cancer is increasing rapidly in targeted studies, there is still no literature on comprehensive analysis of metabolic features and their regulatory mechanisms of different cancer types across multiple ‘omics’ data.In this study, we want to investigate whether different cancer cell lines have distinct metabolic signatures and whether available metabolome data for the NCI-60 cell lines is suitable for cancer subtype classification. We also hope the combined metabolome and transcriptome analysis will reveal some distinct regulatory relationships in some of the NCI-60 dataset which are not otherwise possible by either metabolome or transcriptome study alone. We expect the analysis approach used in this work can be applied to other metabolome-transcriptome datasets when they become available.The first question we would like to address is whether cancer cell lines exhibit tissue-origin specific metabolic signatures. We used classification analysis similar to those performed in microarray data  to classBesides the fact that NCI-60 metabolome dataset has much less data points but with higher noise levels when compared to microarray data, we speculate that the low number of some cancer types (e.g. only 2 prostate cancer cell lines) and high heterogeneity of some cell lines  may also undermine the performance of the metabolome-based classification. In fact, these 4 cell lines contribute most to the out of bag classification error estimates.After prostate, breast, ovarian and CNS cell lines are removed from the whole sample, the metabolite classifiers can reach comparable performance with microarray classifiers Figure . The sigSince the removal of cancer classes will also reduce the complexity of the data thus naturally improve classification performance, we also removed the same number of cancer classes at each stage randomly for 500 times to estimate the effect of improvement of OOB errors due to simplification of data structure resulted from sample removal  essential for cell growth and metabolism. It is likely that although different cancer cell lines may have very different levels of gene expression and metabolism, the steady-state relationship between genes and metabolites in the same or highly coupled pathways should be conserved across different cell lines in the absence of dramatic genomic changes such as gene mutation and copy number variation (CNV). Identification of such gene-metabolite relationships at steady state will help us better understand the underlying molecular mechanisms related to the metabolic change. They will also help us infer potential metabolic changes based on the expression data or vice versa. On the other hand, if a few cell lines deviated significantly from the gene-metabolite relationships exhibited by the most of the cell lines, the related genes in such outlier cell lines are likely to be silenced (no expression) or agitated (over expression) due to genomic structural changes. Consequently, we are interested in both high correlations which reflect steady state trend over all samples, and outliers which reflect signatures in specific cell lines.The classic method of inspecting the relatedness of two quantitative features is by computing the Pearson Correlation Coefficient (PCC). While PCC have been proven to work well in many gene expression studies, the correlation is often inflated by outliers or clustering effect, which made it not suitable to analyze data with high noise level such as metabolic profiles. In contrast, the robust correlations can provide a much better estimate of the true association in the presence of multi-dimensional outliers if the sample size is sufficiently large. We chose the Pair-wise Quadrant Correlation (PQC) for its good computational performance. However, for small sample size, PQC sometimes may inflate the correlation estimate. Therefore our correlation analysis, we use a novel approach by combining results from both PCC and PQC. Gene-metabolite pairs with high PCC and high PQC tend to demonstrate true linear correlation across all cancer types, which imply a general functional association between gene and metabolite profiles. On the other hand, gene-metabolite pairs with high PCC and low PQC are possibly resulted from a few extreme outliers, which can potentially be linked to cell-line specific signatures.Since metabolite-gene relationships can help us to understand the direct or indirect cause of metabolite changes, we computed PCC and PQC for 11872 gene expression profiles and 253 metabolite measurements over 57 cell-lines.To investigate the biological significances of the correlation analysis, we mapped all the known gene-compound associations from EHMN to our NCI60 analysis. There are a total of 721 EHMN annotated direct metabolite-gene associations, involving 352 genes and 81 known metabolites, that overlap with our NCI-60 metabolite-gene relationships.Figure Some of the EHMN gene-compound relationships do show high association across all cell-lines. For example, gene AKR1B1 which reduces L-Arabitol to L-Arabinose with EHMN reaction ID R01758 and R01759, is associated with L-Arabitol with PQC of 0.69 and PCC of 0.36, which implies strong association between metabolite level and gene activity. However, it is surprising that most of the direct enzyme-metabolite relationships cannot be mapped to high correlations Figure  and 2B. Interestingly, the grouping of genes in the same pathway in Figure Further investigation will be worthwhile for high correlation between metabolites and other genes (i.e. those not directly involved in the specific metabolic reaction) in the same pathway, as such un-annotated relationships are likely help us to identify speed limiting enzymes in a pathway, key regulatory genes of related pathways or novel metabolic mechanisms.In our previous analysis we require high PQC in addition to high PCC to identify molecule pairs with true high correlation. We have also identified many cases where cell lines have one or a few gene-metabolite pairs with much higher expression values than the rest of the other samples, which directly produce inflated PCC and very low PQC. To systematically investigate these cases, we used R package mvoulier to detect multidimensional outliers, and recomputed the PCC and PQC scores after outlier removal. Our empirical rule shows that when PCC > 0.6 and PQC < 0.3(Preferably close to 0), and the number of multidimensional outliers is smaller than 3, the high PCC is most likely to be an artifact from very few extreme outliers.To explore the biological significance of these outliers, we compared the outlier cases detected by the criteria mentioned above to the Sanger Cosmic database . Sanger The common feature of these two gene-metabolite pairs is high PCC and low PQC before outlier removal and low PCC and low PQC after the outlier removal. From plots in Figure Since our unbiased analysis did not take advantage of any cell line or gene mutation information, the fact that our top two outliers overlaps with documented gene mutation in the Sanger Cosmic dataset suggests that the mutations are likely to be a cause of such outliers, and the associated metabolites may be good candidate biomarkers for such events.In addition to point mutations, we also compared the outlier analysis with Copy Number Variation (CNV) data from Broad institute. Only 34 out of 57 samples from NCI60 have CNV data, and also found some gene-metabolite outlier pairs that are consistent with CNV outliers. For example, gene BRIP1, with CNV of 14.22 in cell line MCF7, has PCC of 0.90 and PQC of 0.008 and one outlier. The corresponding compound, X-3363, may be associated specifically to this copy number variation.The fact that some top ranked gene-metabolite outlier associations matches with known corresponding genomic structural changes in the specific gene strongly suggests the effectiveness of our approach in identifying distinct molecular processes specific to metabolome and transcriptome variations.http://dtp.nci.nih.gov/index.html March 2007 release). There are 57 cell-lines in 9 cancer classes have both microarray and metabolomics data. We used our in-house Entrez-based Custom CDF version 12 to derive gene-level expression data from the NCI-60 Affymetrix Genechip CEL files (http://brainarray.mbni.med.umich.edu)[NCI-60 data pre-processing: raw molecular datasets were downloaded from DTP web portal (http://www.r-project.org/). Classification and variable selection were performed by R randomForest (http://cran.r-project.org/web/packages/randomForest/index.html) and varSelRF package [http://cran.r-project.org/web/packages/robust/index.html), using the parameter pair-wise Quadrant Correlation (pairwiseQC). Multidimensional outliers were identified by package mvoutlier [All statistical analyses were performed in R  although the small sample size, the high noise level and intra-cancer class heterogeneity in the current NCI-60 metabolome dataset makes it unsuitable for global cancer subtype classification, there are indeed metabolic signatures associated with cancer subtypes. 2) There are biologically meaningful high correlation gene-metabolite pairs across NCI-60 cell lines, identifiable by robust correlation estimates. 3) Most strikingly, there are several examples of abnormal gene-metabolite coupling that can be directly linked to known gene mutations or copy number variations.Conceivably, high correlations as well as outliers can be utilized to aid the progressive prediction of unknown metabolites based on annotations in existing pathway databases and literature. For example, the high correlation of an unknown compound with a known gene and in particular, multiple known genes in a pathway can dramatically reduce the search space for the unknown compound, since the most likely candidates will be structurally related molecules or known metabolites from related pathways. We plan to compare the Mass Spectrometer (MS) features of these unknown compounds from the predicted candidate pool and conduct wet-lab experiments for validation. Since we have discovered that many unknown metabolites are strongly associated with each other but not with any known compounds. Correct determination of even a small fraction of them would facilitate the identification of the rest, which also will in turn improve our understanding of the molecular processes and pathways involving these molecules.In summary, the strong biological relevance of our results also suggest that the analysis strategy we developed based on the combination of PCC and PQC correlation values for identifying real correlation and true outliers is a powerful approach for integrative analysis of noisy ‘omics’ datasets. The presentation of gene-metabolite relationship analysis results in a heatmap grouped by genes in the same pathway together with overlay of known gene-metabolite relationship provides a powerful visual exploration approach for identifying both direct and indirect gene-metabolite relationships. The analysis methods we described here will be useful for other integrated analysis of metabolome and transcriptome and the wet lab validation of novel gene-metabolite relationships.The authors declare that they have no competing interests.GS performed data cleaning, correlation analysis and outlier analysis. CFB helped with correlation analysis and functional inference. CWB cleaned metabolomics data and annotated missing values. GS, BDA evaluated robust correlation methods; FM initialized the study, compiled microarray data and supervised biological validation.


This paper provides a detailed overview and discussion of anaesthesia in patients with mucopolysaccharidosis (MPS), the evaluation of risk factors in these patients and their anaesthetic management, including emergency airway issues. MPS represents a group of rare lysosomal storage disorders associated with an array of clinical manifestations. The high prevalence of airway obstruction and restrictive pulmonary disease in combination with cardiovascular manifestations poses a high anaesthetic risk to these patients. Typical anaesthetic problems include airway obstruction after induction or extubation, intubation difficulties or failure , possible emergency tracheostomy and cardiovascular and cervical spine issues. Because of the high anaesthetic risk, the benefits of a procedure in patients with MPS should always be balanced against the associated risks. Therefore, careful evaluation of anaesthetic risk factors should be made before the procedure, involving evaluation of airways and cardiorespiratory and cervical spine problems. In addition, information on the specific type of MPS, prior history of anaesthesia, presence of cervical instability and range of motion of the temporomandibular joint are important and may be pivotal to prevent complications during anaesthesia. Knowledge of these risk factors allows the anaesthetist to anticipate potential problems that may arise during or after the procedure. Anaesthesia in MPS patients should be preferably done by an experienced (paediatric) anaesthetist, supported by a multidisciplinary team , with access to all necessary equipment and support.The online version of this article (doi:10.1007/s10545-012-9563-1) contains supplementary material, which is available to authorized users. The mucopolysaccharidoses (MPS) are inherited lysosomal storage diseases associated with accumulation of glycosaminoglycans (GAGs) in tissues and organs. They are associated with progressively worsening organ dysfunction that eventually leads to a decreased lifespan. Typical clinical manifestations include coarse facial features, ear–nose–throat (ENT) problems, skeletal dysplasia, growth impairment, cervical instability and spinal cord compression, organomegaly, impaired vision and hearing, joint contractures, hernias and cardiorespiratory disease Muenzer . Some tyN = 544) showed at least one surgical procedure in 75 % of patients, with a median of three to four surgeries per patient  showed surgical interventions in 83.7 % of MPS II patients  pulmonary oedemaComplete airway obstruction (mostly during induction or at extubation): can cause profound hypoxaemia and cardiac arrestPost-intubation problems:StridorLower airway collapse/infectionNeed for reintubation or tracheostomyFollowing is a list of serious anaesthetic complications that may occur during anaesthesia in patients with MPS:The difficulty to ventilate using a face mask or difficulty or inability to intubate  is well illustrated by a case report discussing anaesthesia care in a 10-year-old boy with MPS IH , but these tests are difficult to interpret in MPS patients due to lack of reference data. Standard reference equations based on data from normal individuals may not apply to MPS patients with systemic skeletal dysplasia and extremely short stature. Cooperation problems due to young age, cognitive impairment or behavioural problems may also complicate respiratory function testing. The quality of results obtained by spirometry largely depends on the equipment used and the experience of the operator, particularly in children. A review by a pulmonologist may be very useful preoperatively to optimise respiratory function. The treatment of underlying chronic infection or bronchospasm is important, and preoperative investigation into the severity of OSA can be useful.Spinal cord compression may occur due to spinal canal narrowing at the cervicocranial and thoracolumbar regions in patients with MPS I, II, IV, and VI , requiring emergency tracheostomy  during surgery is required to monitor spinal cord function. This allows identification of surgery- or anaesthesia-induced neurophysiological changes, suggesting changes to the perfusion or direct damage to the spinal cord. Early recognition may prevent permanent damage and neurological deficit. Assessment of MEPs is essential in patients requiring instrumentation at the cervical or lower spine.Neurophysiological monitoring may also be considered in patients undergoing procedures other than spinal surgery, particularly patients at increased risk of spinal cord compression and for long procedures or procedures requiring head movement  patients and exceptional situations Case report from a boy with MPS) IH illustrating intubation difficulties in such patients Case report illustrating preoperative evaluation and anaesthesia in a 15-year old boy with mucopolysaccharidosis (MPS) VI.Fig. 3 LMA laryngeal mask airway (PDF 32 kb)Case report illustrating anaesthetic problems due to airway obstruction and management in a 19-year old girl with mucopolysaccharidosis (MPS) IHS.Below is the link to the electronic supplementary material.


Central acetabular fracture dislocation is usually caused by high-energy external trauma. However, 26 cases that occurred as a result of a seizure attack appeared in the literature from 1970 to 2007, with the seizure attacks themselves caused by many different factors. In this setting, the central acetabular fracture not caused by direct trauma might initially remain unnoticed leading to a delayed diagnosis. In some cases, this may lead to death as a result of massive blood loss. We here present a case of bilateral central acetabular fracture dislocation as a result of a seizure attack. Acetabular fracture dislocations are common and typically result from high-energy external trauma, as observed in patients after high-speed motor vehicle accidents or after a fall with direct impact . HoweverIn this setting, the central acetabular fracture not caused by direct trauma might initially remain unnoticed leading to a delayed diagnosis . In someA 68-year-old Caucasian male was admitted to the internal medicine ward of our hospital for status epilepticus. The patient's medical history included Von Hippel-Lindau disease with multiple cysts in the liver, pancreas, both kidneys and a surgically excised cerebellar hemangioblastoma treated 25 years ago, with the insertion of a Ventriculoperitoneal shunt. He also suffered from epilepsy, recurrent urinary tract infections, chronic renal failure, recurrent atrial flutter, high blood pressure, and Parkinson disease with delirium. His current medication included valproic acid for epilepsy and a combination of trandolapril and verapamil for high blood pressure. In the emergency department (ED) a computed tomography (CT) scan of the head was done and revealed no new abnormalities, and an anteroposterior pelvic X-ray showed a linear nondisplaced fracture of the left acetabulum which remained unnoticed initially . After pAfter two days, the patient was transferred to the internal medicine ward. During this time, the patient was unresponsive, somnolent, or sedated. It was noticed that the patient's blood hemoglobin level was decreasing gradually from 9.8 to 8.1 g/dL over a period of 48 hours. Transfusion of 3 units of concentrated human red blood corpuscles was performed, and his treating internist ordered a CT scan of the abdomen to search for a cause that was thought to be an intracystic hemorrhage. By chance, displaced fractures of both acetabuli with intrapelvic protrusion of both femoral heads were discovered Figures  and 3. ANonoperative treatment was selected because the patient continued to experience mild-to-severe seizures in spite of the antiepileptic drugs. Therefore, bilateral transcondylar traction pins were applied to allow femoral skeletal traction of 8 kg on each lower extremity. The patient remained non-weight-bearing for 3 months. Traction was interrupted at 6 weeks after injury and range of motion exercises of both hips was started and increased gradually. Pain in both hips gradually diminished, and acceptable congruity was achieved based on X-ray findings. Later followup radiography showed the formation of a callus with adequate healing of both acetabuli .Several attempts of full weight bearing with 3 persons support and a walker were unsuccessful at three months, following the injury because the patient developed severe postimmobilization amyotrophy in spite of daily physiotherapy and muscle strengthening exercises. Moreover, in spite of adequate nursing care, the patient developed deep sacral ulcers and died 4 months after his injuries from septic shock. Orthopedic injuries associated with or resulting from convulsions are not uncommon. On rare occasion, tonic-clonic seizure activity has been reported to cause acetabular fracture dislocations –26. UsuaIt is also imperative to mention that long-term seizure patients who are under antiepileptic medications affecting intestinal calcium absorption can suffer from anticonvulsant osteopathy , which mOperative treatment for central acetabular fractures dislocation was reported in some articles , 24. TotThe current case provides an example of a rare and relatively unknown but life-threatening fracture pattern caused by a seizure attack. Late diagnosis of central acetabular fractures may lead to sudden death due to massive blood loss. The mortality rate of such fractures in all reported cases (including our case) is 18.5% (5/27). Hence, the possibility of acetabular fracture dislocation should be kept in mind when examining a postictal patient.


Objective: Although the majority of pharyngocutaneous fistulas close spontaneously with conservative measures, 20% to 30% of patients do not respond to this approach, thereby necessitating major reconstruction with adjacent or free tissue transfers. These procedures carry considerable risk, particularly in patients with medical comorbidities or a history of prior surgery/radiation. Less invasive treatment approaches designed to reverse tissue damage or promote spontaneous healing would represent an important medical advance. Autologous fat grafts have been previously shown to promote healing of persistent wounds and improve the quality of radiation-damaged tissue. In this report, successful closure of a persistent pharyngocutaneous fistula with use of autologous fat grafting is described. Method: The history and details of pharyngocutaneous fistula closure in a patient with recurrent head and neck cancer are reported. Result: A 67-year-old patient with recurrent head and neck cancer and prior radiotherapy underwent reresection including partial pharyngectomy with pectoralis major myocutaneous flap closure. Postoperatively, he developed an enterocutaneous fistula, which failed to close with conservative measures including 8 months of nothing per os. Two rounds of autologous fat grafting were performed with successful fistula healing. Conclusion: Autologous fat grafting is a useful treatment for closure of persistent pharyngocutaneous fistulas. Autologous fat grafting should be considered in poor surgical candidates, particularly in the setting of extensive radiation-induced tissue damage. Although spontaneous resolution ensues in the majority of cases, it can be hindered by factors such as radiation, scarring from previous surgery, and malnutrition.2A 67-year-old white man with a history of hypercholesterolemia, morbid obesity, insulin dependent diabetes, and prostate cancer presented in 2007 with a T2N0M0 squamous carcinoma of the left glottic larynx. Treatment was primary chemoradiotherapy. A local recurrence developed in 2009, which was treated with a left type III, modified radical neck dissection, left tracheoesophageal groove dissection, hemithyroidectomy, laryngectomy, and tracheoesophageal puncture. The postoperative course was complicated by a pharyngocutaneous fistula, which resolved with conservative management. In 2011, a regional recurrence of squamous cell carcinoma developed in the right paratracheal groove with overlying skin involvement. A right radical neck dissection, parastomal skin resection, and partial pharyngectomy was performed. Immediate reconstruction consisted of a right pectoralis major myocutaneous flap to close the hypopharynx defect with split-thickness skin grafting externally. The postoperative course was complicated by a pharyngocutaneous fistula at the superior margin of the permanent tracheostoma . ReirradBased on previous reports demonstrating recovery of radiated tissue with autologous fat grafting, fat transfer was performed in the current patient to improve tissue quality in preparation for flap transfer or as a primary means of achieving closure. Using hand-assisted liposuction techniques, autologous fat was harvested from the patient's abdomen and purified using Telfa rolling. Approximately 8 mL of purified fat were injected into the area immediately surrounding the pharyngocutaneous fistula using both blunt cannulas and 18 gauge needles .9 The ti,13,17Pharyngocutaneous fistulas are a major complication following laryngectomy, reported to occur in 3% to 65% of cases.10,6,18-6,23Studies support that human adipose tissue contains a mesenchymal stem cell population, which may contain regenerative potential.18,26-Greater understanding of ASC biology has led to increased clinical experience with their use both as an ASC isolate and through fat grafting for wound repair. Adipose-derived stem cell injection has led to improved quality and function of damaged tissues in the setting of long-term radiation injury.16The current case report suggests that the use of lipoaspirate transfer may be successful in promoting healing of pharyngocutaneous fistulas. Surgical repair of these problems is difficult necessitating procedures associated with considerable risk and failure; therefore, in complex clinical scenarios fat grafting may be a less invasive and safe treatment option to consider prior to proceeding with further surgery. The complete mechanism of healing in this case is not fully understood although it can be hypothesized that introduction of ASCs via fat grafting is a potential method to restore factors missing for proper wound healing. To better understand the mechanism behind healing using fat grafting, more clinical and basic science experience is needed to fully clarify the beneficial effects and safety of ASCs.