Keratinocyte growth factor (KGF) is a paracrine-acting epithelial mitogen produced by cells of mesenchymal origin, that plays an important role in protecting and repairing epithelial tissues. Pre-clinical data initially demonstrated that a recombinant truncated KGF  could reduce gastrointestinal injury and mortality resulting from a variety of toxic exposures. Furthermore, the use of palifermin in patients with hematological malignancies reduced the incidence and duration of severe oral mucositis experienced after intensive chemoradiotherapy. Based upon these findings, as well as the observation that KGF receptors are expressed in many, if not all, epithelial tissues, pre-clinical studies have been conducted to determine the efficacy of palifermin in protecting different epithelial tissues from toxic injury in an attempt to model various clinical situations in which it might prove to be of benefit in limiting tissue damage. In this article, we review these studies to provide the pre-clinical background for clinical trials that are described in the accompanying article and the rationale for additional clinical applications of palifermin. IntroductionUpdates of KGF basic research– Control of epithelial cell-specific FGFR2b expression– KGF/FGFR2b signalling mechanisms– FGFR2b signalling in tumourigenesis and tumour suppression– KGF family members as pre-synaptic organizing moleculesPre-clinical models of KGF protective and regenerative effects– Palliative care in the oncology setting– Intestinal mucosal injury– Graft-versus-host disease (GVHD) and immune reconstitution– Pulmonary disease– Dermatological applications– Urothelium– PericardiumConclusions and perspectivesvia an exocrine mechanism by virtue of reduced heparin-binding affinity, although still in an FGFR-dependent manner [The fibroblast growth factors (FGFs) comprise a structurally related family of 22 molecules that can be grouped into seven subfamilies on the basis of sequence similarities, conserved chromosomal locations and functional properties . Most FGt manner . Alternat manner , 10.in vitro and in vivo studies have demonstrated that KGF has potent cytoprotective and regenerative effects on epithelial tissues subjected to a variety of toxic exposures [Keratinocyte growth factor (KGF), which was first isolated as an epithelial mitogen from the conditioned medium of human embryonic lung fibroblasts, was the seventh member of the FGF family to be identified, and so is also known as FGF7 , 12. Phyxposures , 36–40. xposures , implyinxposures .For the reasons outlined above, KGF has been the subject of intensive efforts to identify clinical applications in which the integrity of epithelial surfaces is at risk, and the preservation or rapid restoration of these tissues would be of benefit. Currently, a truncated form of recombinant KGF  has been approved for the treatment of severe oral mucositis in patients with hematologic malignancies who have been treated with high doses of chemotherapy and radiation prior to autologous blood progenitor cell transplantation . The purFGFR2 gene, in which exons IIIb and IIIc undergo exclusive and tissue-specific alternative splicing. In this section, we review the mechanisms that act to coordinate the epithelial cell-specific splicing programme.The exquisite specificity of KGF for cells of epithelial origin is determined by alternative splicing of the FGFR2 gene expression, histone marks affect splicing outcome by influencing the recruitment of an adaptor system consisting of H3-K36me3, its binding protein MRG15, and the splicing regulator PTB [As described above, the FGFR2b splice variant, which KGF binds to exclusively , is onlyator PTB .Two paralogous epithelial cell-specific RNA binding proteins, ESRP1 and ESRP2, have been identified as essential regulators of FGFR2 splicing . EctopicA more complete understanding of the signalling mechanisms utilized by KGF to elicit its cytoprotective effects on target epithelial cells will enhance efforts to maximize its pharmaceutical potency, as well as open up the possibility of beneficial combinatorial therapeutic regimens with other pharmaceuticals. In this regard, this section provides an overview of KGF mediated signalling mechanisms.FGFR2b expression system, Luo and colleagues developed a model system to examine phosphotyrosine targets of KGF signalling [By transfecting KGF unresponsive cells with an inducible gnalling . Followignalling  as well gnalling . Phosphognalling , as wellgnalling , 53. Congnalling –59. Sevegnalling . Nonethegnalling , KGF-indgnalling , and KGFgnalling , suggestgnalling , and thegnalling , 64. Fingnalling , 66, andgnalling , 68. Thugnalling .Keratinocyte growth factor induced the antiapoptotic Akt pathway in a mouse model of oxidant-induced lung injury, and inhibition of this activation blocked KGF-mediated protection of the lung epithelium . FGFR2b versus tumour suppression, with particular attention to the specific tissue being targeted.Keratinocyte growth factor is currently approved for use in patients with hematologic malignancies to reduce the incidence and duration of severe oral mucositis that occurs following intensive chemoradiotherapy. If KGF is to be used for a similar purpose in patients with solid tumours of epithelial origin, it must not result in enhanced growth or metastasis, or confer a cytoprotective effect on the malignant cells themselves, rendering them resistant to the therapies designed to kill them. This section reviews the current state of knowledge concerning the impact of FGFR2b signalling on tumourigenesis FGFR2 in endometrial, ovarian, breast, lung and gastric cancer that are thought to promote tumourigenesis. Missense mutations clustered around the third immunoglobulin-like domain result in altered ligand binding specificity and activation of FGFR2 signalling [FGFR2 gene amplification in breast and gastric cancer results in overexpression of FGFR2 and activation of signalling [FGFR2 gene amplification is because of the exclusion of the last exon from the FGFR2 amplicon, and leads to activated FGFR2 signalling based upon constitutive phosphorylation of the FRS2 adaptor molecule [FGFR2 gene are associated with an increased risk of breast cancer [Various genetic alterations occur within gnalling –77. Mutagnalling , 79. C-tmolecule . Five sit cancer –84. The t cancer .Fgfr2b expression show increased sensitivity to chemical carcinogenesis, with the development of papillomas and squamous cell carcinomas bearing oncogenic ha-ras mutations [SPRY4, DUSP4, DUSP6, LRIG1, PHLDA1). Conversely, KGF also down-regulated a set of genes that are expressed at higher levels in SSC cells compared with normal keratinocytes, including a number associated with tumour progression  [A switch from FGFR2b to FGFR2c expression occurs during the progression of bladder, prostate, salivary, and hepatocellular cancer , 86–88, utations . FGFR2b utations . Keratin IGFBP3) . Keratin IGFBP3) , and mic IGFBP3) . Taken tAs reviewed previously, an important question regarding the potential use of KGF to minimize mucositis in patients with tumours of epithelial origin is whether KGF would have a cytoprotective effect on the tumour cells themselves, increasing their resistance to the cytoablative treatments intended to kill them . The avaA recent and hitherto unsuspected role for members of the KGF subfamily of FGFs has been their identification of their involvement in the organization of specific presynaptic terminals in the mammalian brain. Although no preclinical studies have been described in this area, the data discussed below have been included because of their novelty, and because they imply that regulation of these FGFs may have clinical relevance for the prevention of epilepsy or other neurological disorders associated with abnormal synapse formation.in vitro, and are expressed by neuronal subsets in vivo [Axons become specialized for neurotransmitter release at sites of precise contact with their synaptic targets, implying that target-derived factors organize presynaptic differentiation. FGF22 was identified as a major target-derived presynaptic organizing molecule from the mouse brain. FGF22 is expressed by cerebellar granule cells during the period when they receive synapses, whereas FGFR2 is expressed by pontine and vestibular neurons when their axons (mossy fibres) are making synapses on granule cells. Neutralization of FGF22 at post-natal day 3, by injecting into the lateral ventricle of the brain a recombinant fusion protein containing the FGFR2b extracellular domain linked to alkaline phosphatase, inhibited presynaptic differentiation of mossy fibres at sites of contact with granule cells . Conditi in vivo .−/− mice suggests that KGF deficiency impairs inhibitory s formation, resulting in mossy fibre sprouting and enhanced neurogenesis during development, which leads to increased vulnerability to epilepsy [Fgfr2 is inactivated postnatally, the decrease is more pronounced at GABAergic synapses [FGFR2 gene is associated with an increased incidence of schizophrenia, indicating that it may be a potential susceptibility gene for this mental disorder [The differential formation of excitatory (glutamate-mediated) and inhibitory (GABA-mediated) synapses is critical for normal brain function. An imbalance in these synapses may result in various neurological disorders such as autism, schizophrenia, Tourette's syndrome, and epilepsy. FGF22 and KGF are expressed by CA3 pyramidal neurons in the hippocampus. The differentiation of excitatory or inhibitory nerve terminals on dendrites of CA3 pyramidal neurons is specifically impaired in mutants lacking FGF22 or KGF, respectively. Presynaptic defects are rescued by post-synaptic expression of each appropriate FGF. As might be expected from their differential roles in synaptic development, FGF22 deficient mice are resistant to epileptic seizures, whereas KGF null mice are prone to them . Furtherepilepsy . The difepilepsy . Furthersynapses , 113, susynapses  might codisorder .Several preclinical studies have demonstrated the protective effects of palifermin in reducing chemotherapy and radiation-induced gastrointestinal injury and mortality. These striking results have been complemented by investigation of palifermin's cytoprotective and regenerative effects in a number of different tissues and organ systems after exposure to various insults. The findings of these studies are described in the following sections.Preclinical data in multiple animal models of mucositis  providedClinically, adverse events such as rash, erythema and pruritis of the skin and oral cavity are associated with the systemic administration of palifermin . Local dSystemically administered palifermin also increased the proliferation and wet weight of salivary glands in normal and irradiated mice , 122 andDiarrhoea is a complication of abdominal or pelvic radiotherapy and many chemotherapeutic agents, particularly fluoropyrimidines such as 5-fluorouracil (5-FU) and capecitabine, and irinotecan . There aInjury to the bladder also can occur with chemotherapy and abdominal or pelvic radiotherapy. Palifermin pretreatment prevented cyclophosphamide-induced hemorrhagic cystitis in rats  and amelIn summary, the wide array of agents used to treat cancer cause varying types of injury to epithelia in multiple organ systems. Many of these effects are well documented, but as novel targeted agents are approved and used in combination with older agents, new adverse event profiles and dose-limiting toxicities will emerge. Additional preclinical evaluation may be required to determine the therapeutic potential of palifermin in oncology settings where these agents are used.Keratinocyte growth factor is expressed in mesenchymal cells of the intestines including γδ T-cells which play an important role in intestinal growth and integrity. The γδ T-cells express KGF in activated, but not resting states , 128 andThere are a variety of gastrointestinal maladies where loss of intestinal surface area results in malabsorption of fluid, electrolytes and nutrients, and compromise of barrier function. These conditions include IBD, necrotizing enterocolitis, intestinal ischemia/reperfusion, trauma, and bowel resection for tumours and other conditions that leave inadequate length of bowel, known as Short Bowel Syndrome (SBS). Keratinocyte growth factor homeostasis appears to be perturbed in some gastrointestinal disorders, including IBD , 35, 132+CD45RBHi; T-cell transfer) models of IBD, palifermin was able to ameliorate weight loss and improve survival [via oral gavage was also shown to ameliorate acetic acid-induced ulcerative colitis in rats [Most therapeutics for IBD target the immune system. However, many of the symptoms arise from erosion of the intestinal mucosa which leads to intestinal insufficiency and barrier compromise. In acute (DSS: dextran sodium sulphate) and chronic  was preserved by KGF, presumably contributing to maintenance of barrier resistance [Intestinal ischemia/reperfusion injury is encountered in many clinical settings and, like the above conditions, results in loss of intestinal epithelium and increased permeability, sometimes with drastic consequences including sepsis and multiple organ failure . Experimsistance . Studiessistance .et al. [These preclinical data suggest that there is potential for palifermin as a therapeutic for many conditions that result in intestinal insufficiency, and a small study in humans in the oncology setting showed that patients treated with palifermin had retained intestinal barrier function and required less supportive TPN . In humaet al.  found diAllogeneic bone marrow transplantation (BMT) is a common procedure for the treatment of hematologic malignancies. Donor blood marrow and peripheral blood stem cells are routinely used for the reconstitution of immune function in leukaemia and lymphoma patients after radiation and/or chemotherapy. Graft-versus-host-disease is a common complication of allogeneic BMT in which functional immune cells in the donor marrow recognize histocompatibility antigens of the host tissue and mount an immunologic attack. Activated donor T-cells produce an excess of cytopathic molecules, including TNF-α and IFN-γ, which results in tissue damage to the skin, GI tract, liver, lung and immune system.Graft-versus-host-disease can be effectively prevented by depletion of T-cells from the donor, but this results in the loss of graft-versus-leukaemia (GVL) effect, greater risk of engraftment failure, and cancer relapse . Anotherin vivo allo-response, and altered the plasma cytokine levels during acute GVHD. These alterations were manifested by the development of a mixed Th1/Th2 cytokine ratio, in which Th2 cytokines such as Il-4 and Il-13 predominated [Several BMT models have demonstrated a beneficial role for palifermin in limiting GVHD when administered either pre- –160, or ominated –164. Thude novo generation of T-cells in the thymus. The thymus is a specialized organ of the immune system, whose primary function is the production and maturation of T lymphocytes (T-cells) from hematopoietic precursors (thymocytes) derived from the bone marrow. Mature T-cells emigrate from the thymus and constitute the peripheral T-cell repertoire, responsible for directing several aspects of the adaptive immune response. The stock of T lymphocytes is built up early in life, and the function of the thymus diminishes in adults. Involution of the thymus in the elderly is associated with the loss of immune function, susceptibility to infection, and the development of malignancies. The thymic stromal compartment is composed of a complex three-dimensional network of epithelial cells with distinct phenotypes [The outcome of BMT is ultimately dependent upon a successful and comprehensive reconstitution of the immune system with a broad T-cell receptor (TCR) repertoire, which requires the enotypes . Thymic −/− mice have profound defects in thymopoiesis, with diminished thymic cellularity resulting from the impaired proliferation and differentiation of TECs [−/− mice is similar to that of FGFR2b−/− mice, although their thymopoietic effect is less severe [−/− mice exhibit normal thymic organogenesis and post-natal thymopoiesis [via the p53 and NF-κB pathways, resulting in increased transcription of several genes required for TEC function and T-cell development, including bone morphogenic proteins 2 and 4, Wnt5b and Wnt10b [+ thymocytes also produce KGF, which contributes to the expansion of thymic medullary epithelial cells [− T-cells [−/− mice exhibit normal thymic development, they display defective thymic recovery following sublethal radiation, indicating that KGF plays a significant role in post-natal thymic regeneration following injury [Keratinocyte growth factor is expressed within the thymus, both by mesenchymal cells and by T-cells, at specific developmental stages, whereas FGFR2b is expressed by TECs. Embryonic FGFR2b of TECs . The thys severe . In contopoiesis . During opoiesis , 168. Keopoiesis . Keratind Wnt10b . In addial cells . In cont T-cells . Palifer T-cells . Palifer T-cells . Finallyg injury . Such a g injury .+ T-cell dependent mouse model of GVHD directed against minor histocompatibility antigens, it was found that pathogenic donor CD4+ T-cells developed from engrafted hematopoietic stem cells in the stressed and impaired thymus during acute GVHD and mediate the transition to chronic GVHD [de novo generation of pathogenic CD4+ T-cells [+ peripheral blood progenitor transplantation and conditioning with myeloablative total body irradiation, thus providing a primate model for clinically relevant specific immune responses [Palifermin enhances thymopoiesis following experimental BMT by cytoprotection of specific TEC populations within the thymic cortex and medulla, as well as preserving TEC function , 171. ILnic GVHD . Palifer T-cells . These responses .+CD25+Foxp3+ regulatory T-cells (Treg), which are thought to play an important role in the prevention of GVHD [+Foxp3+ Treg numbers via two independent mechanisms [−/− mice, significantly improved engraftment and reduced graft rejection in graft recipients [+ Treg, were used as T-cell providers, the ability of palifermin to improve engraftment was lost [+Foxp3+ Treg-dependent mechanism.Regulatory T-cells are a specialized subpopulation of T-cells that act to suppress activation of the immune system and thereby maintain immune system homeostasis and tolerance to self-antigens. Regulatory T-cells come in many forms, including CD4 of GVHD –179. Palchanisms . The firchanisms . The effcipients . When Scwas lost . Thus, p+ T-cell numbers and function following BMT, various studies have been performed to assess the efficacy of combining palifermin treatment with other promising therapies for GVHD. The tumour suppressor gene, p53, is activated to induce apoptosis and/or growth arrest following genotoxic stress, resulting in the elimination of damaged cells from the organism [in vitro and in vivo [+ and CD8+ T-cells [via chemical castration, when used in conjunction with palifermin treatment, led to the restoration of thymic architecture, number, and subset distribution of TECs [As described above, palifermin facilitates alloengraftment and abrogates GVHD-induced lethality in murine BMT recipients. However, as it fails to completely protect medullary TECs and to restore CD8organism . A small in vivo , abrogat in vivo . Combine T-cells . Androge T-cells –189, and T-cells –195. PreDespite the encouraging results regarding the effect of KGF in reducing GVHD and promoting immune reconstitution in preclinical models, thus far several clinical trials of palifermin conducted in patients undergoing allogeneic stem cell transplants have failed to demonstrate significant advantage in the reduction in GVHD, control of infection or long-term survival (see accompanying review article in this series). Another possible application of palifermin in this general clinical area might be in the setting of hematopoietic stem cell transplants using umbilical cord blood. The use of cord blood stem cells in allogeneic stem cell transplants has increased recently because of the low incidence of GVHD and the fact that they are less immunogenic so that the requirement for HLA matching is less stringent . However−/− mice appeared normal [via a surfactant protein C promoter. Branching morphogenesis was disrupted and the thoracic cavity filled with dilated cystic structures that resulted in embryonic lethality [via regulation of paracrine factors such as VEGF [Keratinocyte growth factor/FGFR2b expression patterns in lung reflect those seen in other epithelial tissues with receptor expression restricted to the tracheal and pulmonary epithelium and the ligand expressed in pulmonary mesenchyme . Althougd normal , KGF hadethality . Howeverethality , 201. Stethality –209 as wethality , 210–212 as VEGF . Keratin as VEGF , 27, 214 as VEGF . PaliferAcute lung injury is a clinical syndrome associated with a variety of conditions including sepsis, pneumonia, aspirations and trauma. It is characterized by injury to the alveolar epithelial barrier, impaired surfactant homeostasis and decreased alveolar epithelial fluid transport  resultinvia suppression of plasminogen activator. Improved survival was observed with IT KGF gene therapy in mice, and correlated with other histological and physiological benefits such as improved arterial oxygen tension [In vitro experiments in the same report suggest that palifermin was providing alveolar protection, rather than enhancing repair, by the prevention of non-apoptotic cell death. Similarly, palifermin improved survival in premature rat pups exposed to hyperoxia. In this model pulmonary hypertension was prevented but pulmonary fibrosis (PF) was not [Keratinocyte growth factor has been evaluated in multiple different models of acute lung injury. In adult rats, where palifermin was administered IT prior to exposure to hyperoxia to induce lung injury, there was a dose-dependent improvement in mortality that correlated with improved lung injury histology . Prolife tension . In neon tension . This su was not .e.g. protein exudation, cellular infiltrates), possibly because of the relatively short time course of the study (1 hr). In models of pneumonia, palifermin restored normal alveolar epithelial fluid transport during the acute phase and lung liquid clearance in early and late-phases, while improving host responses, in part through GM-CSF-stimulated macrophage activation [Other agents also have been used to model ALI. In alpha-naphthylthiourea (ANTU) induced-injury, palifermin attenuated ANTU-induced oedema formation by potentiating alveolar fluid clearance. This effect was mediated through AT2 hyperplasia and increased sodium–potassium-adenosine triphosphatase (NaK-ATPase) activity . In an ativation , 225.ex vivo lung perfusion model of lipopolysaccharide (LPS)-induced injury, KGF present in mesenchymal stem cell conditioned medium improved the rate of alveolar fluid clearance and consequently reduced pulmonary oedema. When the stem cells were pre-treated with KGF siRNA, 80% of the protective effect was abolished, and instillation of KGF alone into the lung lobe partially restored fluid clearance, suggesting that other soluble factors probably contribute to fluid clearance [A few studies have been performed to evaluate the role of KGF in humans with ALI. Endogenous KGF has been detected in pulmonary oedema and BAL fluid from patients with ALI, but its clinical significance is uncertain , 226. Brlearance . Intereslearance . These rlearance  will be Although the beneficial effects of palifermin in animal and human ALI models typically have involved its use prior to the toxic exposure, one case report suggested it might have value when given after the onset of tissue damage . A patievia bone marrow stem cells expressing inducible KGF that home to lung tissue after IV injection [In situ, palifermin reduced edema [Pulmonary fibrosis is characterized by alveolar epithelial cell injury, interstitial inflammation, fibroblast proliferation and collagen accumulation within the lung parenchyma . It can njection . Benefitnjection , decreasnjection , 236 anded edema , neutroped edema  and mitied edema . The effed edema , 238, ined edema  as well ed edema  and hydred edema  content.versus 3 weeks [via IT gene therapy when improvement appeared to be mediated by antioxidant activity, as well as up-regulation of surfactant proteins and down-regulation of TGF-beta [The development of PF in response to radiation has a longer time course compared with bleomycin instillation (12 weeks  3 weeks ). A sing 3 weeks . In expe 3 weeks , and viaTGF-beta .via proliferative effects.Keratinocyte growth factor therapy has been evaluated in lung transplant models with mixed success. Palifermin treatment of isogenic donor rats prevented intra-alveolar oedema, which was attributable to increased production of surfactant protein C and corresponding improvement in surfactant function . These min vivo and in vitro [The utility of KGF has also been examined in models of other pulmonary diseases. Emphysema is a chronic obstructive pulmonary disease, largely attributed to cigarette smoking, featuring an abnormal dilation of distal airspaces combined with destruction of alveolar walls . In a moin vitro .Taken together the studies discussed above comprise a large body of evidence that palifermin may have therapeutic potential for a variety of human pulmonary disorders. Most animal studies indicated that palifermin's benefits are dependent on pre-injury dosing that presumably creates a functional tissue reserve to facilitate protection, although some studies revealed shorter term cellular responses to palifermin that also may have clinical benefit. Typically the onset of human lung injury cannot be anticipated, except in the setting of cancer therapy or lung transplantation. This might limit the practical application of palifermin. However, Ware and Matthay  point ouThe observations that KGF was dramatically up-regulated following cutaneous injury in mouse and human full-thickness wounds , 31 as wBased upon such observations, experiments were performed with KGF in animals to determine whether its topical application to the skin could stimulate epidermal wound repair. Keratinocyte growth factor increased the rate of re-epithelialization and epidermal thickness in partial-thickness wounds of the porcine epidermis  and wounAloe vera is a succulent xerophtye in which the innermost part of the leaf consists of large, thin-walled parenchyma cells that retain water in the form of viscous mucilage for storage purposes, and which has long been used for its therapeutic properties. The use of cell wall fragments derived from this gel to bind KGF in microsheets has been described for wound healing applications, although the efficacy of this technique has not been reported [A biomimetic approach has been used to investigate cell-controlled delivery of KGF. For this purpose, a matrix-binding peptide that contains two domains, one recognized by the transglutaminase factor XIII and the other containing the fibrin binding site of α2-plasmin inhibitor, was covalently attached to free amines on the surface of the KGF molecule. Upon activation by thrombin, factor XIII acts on fibrin to form γ-glutamyl-ε-lysyl amide cross-links between fibrin molecules to form an insoluble clot. Thus, during coagulation, the transglutaminase activity of factor XIII recognizes one domain of the hybrid molecule, resulting in its incorporation into the fibrin matrix at high concentration. Subsequent, cell-mediated activation of plasminogen to plasmin degrades the fibrin matrix and cleaves the hybrid peptide at the fibrin binding site to release a modified, yet still biologically active, KGF molecule into the local microenvironment . Treatmereported .The FGF binding protein (FGF-BP), originally shown to bind and activate FGF1 and FGF2, also interacts with KGF, FGF10 and FGF22, and enhances the activity of low growth factor concentrations. Furthermore, expression of FGF-BP is increased following injury to murine skin, particularly in keratinocytes . Thus, uin vivo cell targeting as well as direct patient exposure to viral vectors, and offers the possibility of delivering biologically active proteins in a sustained manner to improve the chance of therapeutic success. A similar approach involved the use of HaCaT keratinocytes stably expressing high levels of KGF to enhance the healing of superficial second degree burns generated on pig skin, and indicated that engraftment of KGF expressing HaCaT cells significantly reduced the time required for complete re-epithelialization compared to control cells [Several methods have been developed for gene transfer. In rats with a full thickness scald burn, subcutaneous injection of a KGF cDNA liposomal complex resulted in significantly improved epidermal regeneration, because of increased keratinocyte proliferation and decreased apoptosis . Liposomol cells .LAMB3 gene (coding for the beta-3 subunit of laminin), resulting in small and sparse hemidesmosomes [Epidermolysis bullosa (EB) is one of several types of skin-blistering disorders with considerable morbidity and mortality. Although EB is a clinically and genetically heterogeneous syndrome, it is now categorized into four main subtypes according to the level of skin cleavage: EB simplex, junctional EB, dystrophic EB and Kindler Syndrome . Specifismosomes . The gensmosomes . A variesmosomes , with eajeb mutant mice have reduced levels of LAMC2 protein and develop a progressive blistering disease that closely resembles a sub-type of junctional EB  [The epithelial growth and reparative effects of KGF seem to be suited for therapy in the EB settings where the overall stimulation of epidermal keratinocyte proliferation and subsequent thickening of the epithelial layer might provide some protection from injury and/or aid in repair of skin and mucosal lesions. In a porcine model of epithelial wound healing, KGF increased the number of serrated basal cells associated with increased deposition of collagen fibres in the superficial dermis. Electron microscopy documented better developed hemidesmosomes and thicker bundles of tonofilaments in the serrated cells . In miceHerlitz) .The rational for testing of palifermin in models of dystrophic EB is more complicated. The dermal fibroblasts would not respond to palifermin therapy, at least directly, as they do not express the KGF receptor. Early clinical data have shown that stem cell transplantation is a promising therapy for EB but the risk/benefit ratio of the conditioning regimen, which relies on immunomyeloablative chemotherapy, was such that it was only used in severe disease of recessive dystrophic EB . AlthougOne of the most common complications of EB is the occurrence of skin cancer . Althoug−/− mice, the developing ureteric bud and mature renal collecting system were markedly smaller than in kidneys from wild-type mice, and mature kidneys in KGF−/− mice had 30% fewer nephrons than wild-type kidneys [In vitro experiments demonstrated that KGF augmented ureteric bud growth and increased the number of nephrons that formed in rodent metanephric kidney organ cultures [−/− mice was markedly thinner than that of wild-type mice, and lacked the intermediate layers present in normal animals [The urothelium comprises the epithelial lining of much of the urinary tract, including the kidneys and bladder. In KGF kidneys . In vitrcultures . Conditicultures . The bla animals . Primary animals . Systemi animals . Rhesus  animals .S--L-cysteine (TFEC). The FGFR2b transcript level was high in the papilla and medulla of the undamaged kidney, but increased further following TFEC administration [in vivo administration to control mice, whereas inhibition of FGFR2 by an antisense oligonucleotide inhibited urothelial proliferation following IRI. Furthermore, IRI resulted in FGFR2 phosphorylation, as well as up-regulation of KGF [Keratinocyte growth factor mRNA expression increased several fold following surgical bladder injury in rats , indicatstration . These rn of KGF . The FGFn of KGF .Palifermin pre-treatment almost completely ameliorated cyclosphosphamide (CP)-induced ulcerative hemorrhagic cystitis in rats, with the protective effect thought to be because of the rapid proliferation of urothelial cells during and after chemotoxic injury, as well as cytoprotective mechanisms . AdminisThe pericardium is a double-walled sac that contains the heart and the roots of the great vessels. Pericardial adhesions, which represent an attachment of the pericardium to the heart muscle, can form after surgical procedures, trauma or inflammation and may restrict the action of the heart. Injury to mesothelial cells, which comprise the membrane that lines the pericardium, impairs the fibrinolytic capabilities of the mesothelial membrane and promotes adhesion formation . Keratinvia multiple mechanisms, including the stimulation of cell proliferation, migration, differentiation, survival, DNA repair and the induction of enzymes involved in the detoxification of reactive oxygen species [Keratinocyte growth factor possesses many attributes required of a molecule that plays an important, if not crucial, role in mediating epithelial tissue repair following injury. It is up-regulated following tissue injury, and it acts to strengthen the integrity of the epithelial barrier  species . SubsequPre-clinical studies have explored multiple areas of application. The largest effort has focused on palliative care in the oncology setting, as it was postulated that palifermin could protect tissues from the harmful effects of chemoradiotherapy regimens, thus limiting the toxicities associated with their use, such as mucositis, xerostomia and diarrhoea. The advantages of pursuing such a course were obvious as the timing of palifermin treatment relative to the toxic exposure could be readily controlled. This was particularly appropriate as the beneficial effects of palifermin typically are most pronounced when it is administered prior to the noxious agent. Other studies have tested the effects of palifermin in the context of acute organ injury such as ALI. Clearly, in these applications, palifermin cannot be administered before injury occurs, and so the strategy is dependent upon the ability of palifermin to limit the extent of tissue damage after injury and/or to accelerate repair processes. Pre-clinical studies suggest that palifermin might have utility as a biodefense therapeutic for public and/or emergency personnel to mitigate radiation syndromes in situations when radioactive material has been released.In some cases, pre-clinical studies have provided important new insights into KGF function, and in doing so have suggested new avenues of investigation to be pursued. For example, it was originally proposed that palifermin might limit GVHD by reducing injury to the GI tract that results from chemoradiotherapy conditioning regimens prior to BMT. While several BMT models demonstrated a beneficial role for palifermin in reducing GVHD, subsequent studies indicated an immunomodulatory mechanism of palifermin that is in addition to, and independent of, its ability to limit the damage to epithelial tissues caused by conditioning regimens.The accepted paradigm of KGF functioning as a mesenchymal mediator of epithelial growth and regeneration was challenged by the observations that KGF family members act as pre-synaptic organizing molecules. The differential formation of excitatory (glutamate-mediated) and inhibitory (GABA-mediated) synapses is critical for normal brain function, and an imbalance of these synapses may play a role in the pathogenesis of various neurological disorders associated with abnormal synapse formation such as epilepsy, autism, schizophrenia and Tourette's syndrome. These studies demonstrate an unexpected new area for potential clinical development of palifermin.The results described in this review document the remarkable ability of palifermin to protect or facilitate the repair of many different epithelial tissues following injury in pre-clinical models. The goal of such studies was to provide supporting evidence for efficacy in corresponding clinical situations. The accompanying article in this series reviews current information about palifermin use in a number of clinical trials primarily involving palliative care in the cancer setting. While the findings of some clinical trials have borne out the positive results from pre-clinical studies, others have been less convincing (see accompanying review article in this series). Such inconsistencies between pre-clinical models and clinical studies probably reflect inherent differences between animal and human biology, as well as sub-optimal dosing regimens that will require further adjustment. Furthermore, palifermin might be used in combination with other pharmaceutical agents in a particular setting to maximize its beneficial effects. Nonetheless, animal models have been invaluable in demonstrating the pharmaceutical potential of palifermin, and will likely continue to be of considerable importance in promoting further clinical development.


Biomaterial-related infections are a persistent burden on patient health, recovery, mortality and healthcare budgets. Self-assembled antimicrobial peptides have evolved from the area of antimicrobial peptides. Peptides serve as important weapons in nature, and increasingly medicine, for combating microbial infection and biofilms. Self-assembled peptides harness a “bottom-up” approach, whereby the primary peptide sequence may be modified with natural and unnatural amino acids to produce an inherently antimicrobial hydrogel. Gelation may be tailored to occur in the presence of physiological and infective indicators  and therefore allow local, targeted antimicrobial therapy at the site of infection. Peptides demonstrate inherent biocompatibility, antimicrobial activity, biodegradability and numerous functional groups. They are therefore prime candidates for the production of polymeric molecules that have the potential to be conjugated to biomaterials with precision. Non-native chemistries and functional groups are easily incorporated into the peptide backbone allowing peptide hydrogels to be tailored to specific functional requirements. This article reviews an area of increasing interest, namely self-assembled peptides and their potential therapeutic applications as innovative hydrogels and biomaterials in the prevention of biofilm-related infection. Biomaterials have an increasingly important role in patient care. Approximately five million medical devices and implants are used in the US each year . They arGrainger and colleagues researched the contribution medical devices have to improving patients’ quality of life . They coStaphylococcus epidermidis, specifically recognize binding sites and mediate adherence to the surface via covalent attachment.Biofilms are phenotypically heterogeneous, sessile microbial communities that form at biotic and abiotic sites. They may be composed of a single or multiple species  of bacteria and fungi ,11,12. B®, also suffer from microbial contamination due to mechanical rather than chemical factors [Staphylococcus epidermidis [in vivo. Similar research performed by the Gottenbos group suggested that biofilm formation is a complex relationship involving not only the biomaterial surface properties and the ability of the bacteria to bind to a given surface, but also the availability of nutrients at the site of adherence [The potential for microbial spoilage is not confined to hydrophobic surfaces. More hydrophilic materials, for example Teflon factors . MacKintdermidis . They dedherence .Staphylococcus aureus and Candida species [Accumulation on a surface occurs when bacterial cells multiply and form multi-layered cell clusters resulting in a complex three dimensional architecture- the classic biofilm . The bio species . Removal species ,21,22.Escherichia coli, Proteus mirabilis, Pseudomonas, and Candida [Typical biofilm related medical device infections include: Catheter associated urinary tract infections; peristomal skin infections following insertion of percutaneous endoscopic gastrostomy feeding tubes; and pneumonia or tracheobronchitis with tracheostomy devices. The most commonly implicated pathogens are: staphylococci, enterococci,  Candida ,23,24. TNumerous strategies to reduce biomaterial-associated infections have been developed but few have translated to clinical practice . HospitaBiomaterials cover a diverse range of pharmaceutical applications from drug delivery to tissue engineering . Every d® are both coated by a combination of minocycline and rifampicin [Candida albicans and Escherichia coli for up to seven days [Staphylococcus epidermidis (ATCC 35984) and Staphylococcus aureus (ATCC 29213) but for an extended period of up to 21 days [ica operon expression [Current research has seen an increased focus on the antimicrobial properties of silver. Hydrogel/silver coated urinary catheters have been investigated and promising results obtained. A reduction in the primary bacterial adherence was observed in comparison to a standard silicone catheter indicating that hydrogel/silver coatings have the potential to delay the onset of catheter associated infections . Synergifampicin . Silver ven days . Minocyc 21 days . A range 21 days . Sub-inhpression , linked pression ,40, occu®, suffer from a reduction in activity due to masking of antimicrobials by the host’s conditioning film [One of the major limitations regarding the delivery of antimicrobials in a biomaterial model is the effect of burst-release. Burst-release is consistent with an initial high and rapid release of the antimicrobial from the biomaterial. It is one of the major challenges of modern drug delivery. The antimicrobial reservoir depletes to sub-inhibitory levels within days allowing infection to develop unchallenged. Covalent attachment of antimicrobial, as outlined by the examples of ARROWgard™, BioGuard Spectrum™ and Cook Spectruming film . A signiing film . A multiing film . The autThere has also been increasing interest in using peptides for biomaterial applications . As antiThe process of self-assembly is an important parameter for the development of novel biomaterials. It is particularly relevant with regards to nanotechnology. These principles have been adopted from nature. Ribosomes and the quaternary haemoglobin structures are examples of naturally occurring self-assembled architectures . Assembl® patch [Candida albicans biofilm formation relative to untreated polystyrene controls [Staphylococcus aureus and Staphylococcus epidermidis adherence to polymethylmethacrylate and increased susceptibility to vancomycin and gentamicin was also observed in the presence of poloxamer 407 [Staphylococcus aureus and a Pseudomonas aeruginosa strain isolated from cystic fibrosis patients. Pluronics have also been linked with increased wound-healing in animal models validating continuous research into their applications as effective biomaterials [Current polymer technologies include the area of Pluronics or poloxamers. Composed of three polymers polyoxyethylene (PEO), polyoxypropylene (PPO) and polyoxyethylene (PEO), Pluronics is an area that has been extensively studied with regard to micellization and the formation of structures for drug delivery purposes. Pluronics have the ability to assemble at physiological temperatures, in a variety of solvents, and over a range of concentrations as opposed to a single critical concentration . There i® patch . The pos® patch . Wesenbecontrols . Reductiamer 407 . These samer 407 . In thisaterials .Pseudomonas aeruginosa biofilm over a three day period. Self-assembled monolayers have been created to modify the surface properties of biomaterials with the aim of reducing bacterial attachment. Recently Kruszewski et al. produced stainless steel, commonly employed in orthopaedic implants, modified by a self-assembled monolayer of long alkyl chains terminated with hydrophobic (−CH3) or hydrophilic (oligoethylene glycol) tail groups [Staphylococcus aureus biofilm formation for up to 24 and 48 h respectively. Protein deposition by the host conditioning film remains a problem due to masking of covalently attached molecules. Studies exist for self-assembled monolayer materials that display resistance to biofilm formation and host protein adsorption. Self-assembled monolayers of alkanethiols, presenting a tri(ethylene glycol) functional group, displayed a profile of reduced protein adsorption and mammalian cell adhesion and resisted Escherichia coli biofilm formation [co-acrylic acid proved resistant to adhesion by Staphylococcus epidermidis for up to 10 h [Related research combines innovative approaches to stimulate antimicrobial release with self-assembled hydrogel polymers. Norris and colleagues synthesized a self-assembled poly(2-hydroxyethyl methacrylate) coating containing ordered methylene chains . This pol groups . These gormation . The aut to 10 h . LoadingSuch approaches have been significant in advancing biofilm-resistant self-assembled polymer research. Limitations exist affecting translation into clinical practice, namely sufficient antimicrobial action over a sustained period of time. Self-assembled polymers have contributed to a multitude of innovative investigations and applications within biomedical engineering. It is now a widely accepted method of material development. The theory has been expanded to investigate the potential of peptide self-assembly and peptide-based nanomaterials. Self-assembled peptides display great promise as antimicrobial coatings. Alteration of the peptide backbone allows materials to be tailored to their application. The production of an inherently antimicrobial peptide hydrogel, which self-assembles in response to environmental or specific pathogenic stimuli, may allow for significant reduction of biofilm formation over a prolonged period.Uses for self-assembled peptides range from medical to engineering applications. They have recently come to the fore of bioscience contributing significantly to the nanotech revolution . Work by2) can also contribute to assembly via hydrogen bond intermolecular interactions with surrounding solvent and neighboring peptide molecules. Charged amino acids, for example lysine, result in charge-charge electrostatic interactions. These have potential to drive or prevent assembly depending on the degree of polarity and charge density. Salt concentration is also a contributing factor, as demonstrated through work on the peptide EAK16-II by Hong and colleagues [Ka, has a large influence on the self-assembly process. The natural amino acid tyrosine allows further functionalization of peptide molecules. Its R-group consists of an amphiphilic phenol grouping which allows, for example, formation of imines under relatively mild conditions (pH ~6.5) [Peptides are ideal building blocks for the formation of nanostructures. Utilizing a “bottom up” approach, individual amino acid residues are used to build higher ordered structures . The twelleagues . A low spH ~6.5) . The usepH ~6.5) .Peptide derivatives as detailed include amphiphiles and π-stacked aromatic peptides. Amphiphiles have a similar structure to membrane phospholipids, possessing a hydrophobic alkyl tail conjugated to a charged moiety. They are ideal molecules for interacting with and disrupting bacterial membranes . AssemblReviews such as those by Gazit and Martinez ,85 proviin situ [Although the aim of assembly is to achieve higher-ordered structures, these must be uniform, homogenous and reproducible with defined and useful properties. Peptides provide an ideal basis for such structures for many reasons. They are biocompatible and biodegradable . Their sin situ . Peptidein situ . They deThe chemical, physical and structural properties that govern peptide self-assembly are closely associated with factors that determine the antimicrobial potency and spectrum of activity of antimicrobial peptides. Antimicrobial peptides occur throughout nature and are involved in the innate immune response. This response is mounted a short time following exposure to an infective agent and stimulation of cells known as Toll-like receptors . Unlike The amphiphilicity of antimicrobial peptides allows them to target microbial cell membranes, particularly those of bacterial cells. Bacterial membranes possess an overall negative charge, compared with neutrally charged eukaryotic membranes, due to the presence of acidic hydroxylated phospholipids. These include cardiolipin, phosphatidylglycerol and phosphatidylserine . The preCationic peptides are the most studied form of antimicrobial peptides. Possessing a net positive charge, they are most commonly composed of 12–50 amino acids, approximately 50% of which are hydrophobic . Cationil-stereoisomer form. Stereoisomerism is based on the ability of molecules to rotate plane-polarized light. l-amino acids are easily recognized by host and bacterial enzymes. They are ideal substrates for proteolysis by peptide specific enzymes termed proteases. In theory this limitation can be reduced, to increase both therapeutic efficacy and retention within the host, by replacement with d-amino acids. Amino acids that are in the d-enantiomeric form do not fit in the active site of proteases and therefore cannot be broken down. d-amino acids do have limitations. They are typically expensive, and for short peptides the realistic enzyme stability they offer is debatable [Antimicrobial activity and self-assembly of peptides are influenced by similar factors namely: charge, bulk and lipophilicity. Strøm and co-workers investigated the contribution of these factors to antimicrobial activity . Their febatable .® Outcomes Registry and Experience (CORE) 2004 database. This covered patients administered with the injectable form of daptomycin (brand name: Cubicin®) for the treatment of infective endocarditis [Staphylococcus aureus infections were methicillin resistant, and 43% enterococci infections were vancomycin resistant. The findings showed that daptomycin has a potential role in treating these types of infections, with 63% of treatments proving successful overall. Similarly a review by Li highlights in great detail the effective role of polymyxin E (colistin) via inhalation and intravenous administration to treat multi-resistant pseudomonal infections in cystic fibrosis patients [Lipopeptides are a form of antimicrobial peptides present in nature among bacteria and provide selective advantage against competing microbial strains. Their success is reflected in the pharmaceutical industry, with antimicrobial lipopeptides already successfully utilized clinically. These include anionic daptomycin , which fcarditis . Of thospatients . There ipatients .et al. to act synergistically with rifampicin against multi-drug resistant Pseudomonas aeruginosa in both in vivo and in vitro models [Stenotrophomonas maltophilia isolated from cystic fibrosis patients [Escherichia coli and 36% Pseudomonas aeruginosa displayed resistance to fluoroquinolones [Research is increasingly focused on the action of antimicrobial peptides against resistant biofilm phenotypes . Evidenco models . The lippatients . Infectiinolones . There iinolones . The areinolones  and our inolones .et al. to produce a series of ultrashort cationic lipopeptides [12-ornithine-ornithine-tryptophan-tryptophan displayed the most potent activity particularly against Gram-positive bacteria with complete eradication obtained within 24 h exposure against mature biofilms of Staphylococcus epidermidis (ATCC 35984) at concentrations as low as 15 µg/mL [Ultrashort refers to peptides up to 7 amino acids in length. They have become increasingly attractive in the development of peptide therapeutics due to reduction in associated cost and ease of synthesis. Amino acids are rationally selected in line with a minimum pharmacophore required to permit antimicrobial activity . Residuepeptides . Based o15 µg/mL . The gro15 µg/mL .An alternative approach is to tag the end of the peptide residues with hydrophobic, bulky aromatics such as phenylalanine and tryptophan. Work on this area has involved ultrashort peptides composed of 4–7 amino acid residues. As with lipopeptides, these compounds are amphiphilic in nature and readily interact with target cell membranes. Of the tagged peptides investigated by Pasupuleti and colleagues , the mosin vivo highlighting their clinical potential. Livne demonstrated OAKs incorporating synergistic erythromycin had improved survival rates in neutropenic mice infected with multi-drug resistant Escherichia coli compared to erythromycin or OAK monotherapy [in vivo erythromycin efficacy. Cochleates are a drug delivery vehicle consisting of liposome-like molecules formed by a lipid-based supramolecular assembly of natural products (phosphatidylserine), negatively charged phospholipid and a divalent cation . The work of Sarig underlines the importance of the drug delivery vehicle in ensuring promising in vitro results for antimicrobial peptide-like molecules are translated efficiently to clinical practice. This is the greatest challenge in drug development and is particularly difficult with regard to peptides and proteins which suffer from poor solubility, pharmacokinetic properties, stability and antigenicity issues [Oligo-acyl-lysines (OAKs) are peptidomimetic molecules that mimic the primary structure and function of naturally occurring peptides. They are composed of acyl-lysines, with the acyl chain length determining hydrophobicity of the molecule and lysine conferring cationic charge for targeting of bacterial membranes or inhibition of cellular processes linked to intracellular DNA . They caotherapy . Sarig dotherapy . Housingy issues . Hence ay issues .The development of peptides that self-assemble upon exposure to environmental stimuli is of increasing interest in biomedicine. The ability to modify a peptide to assemble on cue provides the peptide with a range of desirable properties and potential applications including targeted drug delivery in the area of antimicrobials. For biomaterials, preventing the formation of a highly resistant biofilm phenotype is pivotal in preventing infection throughout the life of the material . HarnessKa of the peptide, the amino acid backbone and substituent functional groups. The effect of salt on pH-triggered assembly is also an important factor in determining the degree of ionic interactions [DPPT peptide motif  [Ka of lysine’s primary amine R-group (pH > 9) resulted in self-assembly as intramolecular folding resulted in a hydrophobic valine core surrounded by a hydrophilic lysine surface. Lowering the pH, below the pKa of lysine favored charge repulsion and disassembly. Addition of buffered saline (150 millimolar sodium chloride) or Dulbecco’s Modified Eagle’s Medium (165 millimolar sodium chloride) to 2% w/v of MAX-1 in water at pH 7.4 directed the formation of a biocompatible hydrogel. Formation of a hydrogel was due to screening of positive charged lysine moieties by chloride ions and alteration of the hydrophobic: charge balance allowing a decrease in solubility, therefore promoting assembly [pH and ionic strength are the most commonly utilized methods to achieve assembly and disassembly of peptides on cue. Charge interactions are crucial to the assembly process and are influenced by the practions . The Schreonine) . Lysine assembly .Staphylococcus epidermidis, Staphylococcus aureus and Streptococcus pyogenes) and Gram-negative (Klebsiella pneumoniae and Escherichia coli) bacteria [Staphylococcus aureus. A second peptide molecule, PEP6R, consisting of six arginines within its peptide primary structure, demonstrated broad spectrum activity against Staphylococcus aureus, Escherichia coli and multi-drug resistant Pseudomonas aeruginosa. Both were able to form hydrogels at physiological pH.Replacing lysine with aspartic acid at amino acid position 15 (MAX-8) lowered the overall charge of the molecule by +2 and was sufficient to decrease gelation time from 30 min (for MAX-1) to 1 min . Encapsubacteria . The resbacteria . MARG1, Escherichia coli and Staphylococcus aureus. Gels containing only two arginine residues demonstrated limited activity against Gram-negative Escherichia coli. Peptide derivatives were more active against Gram-positive bacteria. The presence of divalent ions such as calcium (Ca2+) reduced the peptides antimicrobial activity. The group concluded that in order to achieve optimal antimicrobial activity a minimum of four arginine residues were required.A related arginine rich peptide PEP8R (containing 8 arginine residues) was also created . Variati3-NH2 species conjugated to a central tetrapeptide linker (TDPPG) [Escherichia coli after 36 h incubation. High bacterial loads (greater than 107 colony forming units per mL) resulted in loss of inhibitory capacity. Again membrane disruption due to the presence of polycationic lysine residues was the most plausible mechanism for antibacterial activity.Recent research by Liu and colleagues demonstrated the rational design of a pH dependent self-assembled antimicrobial peptide (ASCP1) consisting of two peripheral (KIGAKI) (TDPPG) . SimilarBacillus subtilis and Staphylococcus aureus) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria, with minimum inhibitory concentrations determined. Compounds with the most broad-spectrum antimicrobial effect had hydrophobic phenylalanine closely attached to the Fmoc moiety. The close proximity of fluorenyl and phenyl moieties allowed an optimum hydrophobicity, described by the authors as the “threshold hydrophobicity”, to be obtained thus allowing significant activity against bacteria via membrane attack.RADA16 is one of the most comprehensively studied self-assembled peptides due to its ability to self-assemble at physiological pH . MarketeProteus mirabilis. Urease catalyses the breakdown of urea to ammonia via hydrolysis leading to increased pH at the urine-catheter interface and the precipitation of mineral salts such as calcium phosphate (hydroxyapatite) and magnesium ammonium phosphate (struvite). A combination of encrustation and biofilm formation leads to blockage of the catheter, with removal of the device necessary to resolve infection [Proteus mirabilis.pH triggered self-assembly has potential to be utilized in antimicrobial therapies. Urinary catheter associated infections are synonymous with an increase in alkaline conditions due to the presence of the enzyme urease synthesized by Gram-negative nfection . We hypoN-methylpyridyl)porphyrin), which binds electrostatically with methacrylate groups of a methacrylic acid or a methyl methacrylate copolymer [Staphylococcus aureus and Pseudomonas aeruginosa.Light may be used as an environmental trigger for the formation of hydrogels. These are typically formed when a water-soluble polymer and photo-initiator are exposed to a specific wavelength of light and crosslinking occurs. The resulting hydrogels display temporal and spatial resolution. Macromolecules are the preferred starting material and these are chosen to achieve the desired crosslinking and mechanical properties for the final hydrogel. The photo-initiator must be sufficiently reactive yet cytocompatible. Exposure to ultraviolet light triggers arrangement of a peptide into β-hairpins and subsequent self-assembly to a hydrogel occurs provided that the side chains permit this. Cysteine is hydrophobic in nature and easily functionalized. Therefore it is a useful side chain component for facilitating assembly. An example of this type of assembly is the 20-residue MAX7CNB peptide . This caopolymer . VisibleKa demonstrated self-assembly had occurred at 25 °C but only sufficient to produce a viscous solution. Gelation occurred between 55 and 80 °C which the authors attributed to dissolution of precipitate, formed due to a low pH, allowing homogenous hydrogels to be formed in solution. Further work by Tang studied glycine and leucine substituted Fmoc dipeptides [Temperature can be used as a trigger to form higher assemblies (hydrogel structures) either through cooling or heating of molecules. The degree of hydrophobicity governs the temperature at which folding occurs. Typically, assembly of more hydrophobic molecules takes place at a lower temperature . Schneidpeptides . In addiEnzyme mediated self-assembly occurs as a result of catalysis or removal of a blocking group within the peptide primary sequence. It occurs under standard physiological conditions (pH and temperature) . EnzymesEscherichia coli, that overexpressed a phosphatase enzyme, was able to be selectively inhibited by a short naphthalene containing peptide. Dephosphorylation of soluble phosphorylated NapFFY(p), NapDFDFY(p) and Nap-β3-HPhg-β3-HPhgY(p)  occurred in the cell cytoplasm causing inhibition of multiple intracellular processes resulting in a reduction in bacterial viability. Further work by the Xu group demonstrated how peptide self-assembly can be governed by a tyrosine-linked kinase/phosphatase switch [in vivo in the presence of phosphatase enzyme, as removal of hydrophilic phosphate groupings increased the hydrophobicity of the molecule, driving self-assembly. The presence of phosphatases have been attributed to medical device related pathogens such as Escherichia coli, where alkaline phosphatase is present in the periplasmic space and therefore serves as a valid microbial target [Escherichia coli. This mechanism has potential to be exploited for the detection of extended spectrum β-lactamases in a clinical setting and allowing screening of potential inhibitors.As outlined previously , selectie switch . Phosphol target ,150. Thel target  also pro31P NMR) allowed determination of the rate of dephosphorylation and inhibition within defined areas of the bacterial cell and enabled the location of formed fibers to be identified. Treatment with alkaline phosphatase facilitates protonation of phosphate groups and the formation of higher assemblies. Peptide hydrogels formed over 24 h. Assembly was evident at a molecular level and there was evidence of β sheets, hydrogen bonding and π-stacking. Ability to assemble in vivo was investigated using media with and without the nucleoside inosine, which increases alkaline phosphatase synthesis two-fold. Bacterial cells were treated with a FmocFYp-OH precursor and HPLC analysis showed that the peptide moved into the hydrophobic environment of the bacterial cells. This demonstrated that assembly could occur in vivo. Treatment of Escherichia coli cells with other precursors showed similar results (self-assembly) however the effect of hydrophobicity on assembly and the resulting location of the formed peptides varied. More hydrophilic peptides were more likely to partition into the surrounding media rather than remaining within the hydrophobic environment of the bacterial cell. The findings indicated that formation of the peptides followed by movement out of the bacterial cells was sufficient to significantly reduce the number of viable bacterial cells. Therefore retention of the formed nanostructure within the cells was not essential to exert an antimicrobial effect. The intracellular and extracellular modes of action of these peptides reduce the ability of bacteria to develop resistance and bode well for their future development as therapeutics. The Ulijn group also employed the esterase subtilisin, obtained from the soil derived bacterium Bacillus licheniformis, as a mediator for assembly of Fmoc dipeptide methyl esters proving the versatility of the enzymatic approach to allow self-assembly [Enzymatic assembly of ultrashort peptides, utilizing alkaline phosphatase, has been conducted more recently by the Ulijn group . The Fmoassembly .et al., produced a macroscopic tripeptide hydrogel (DLFF) at physiological pH which incorporated the sparingly soluble antibiotic ciprofloxacin [in vitro. Mild antibiofilm activity was demonstrated for the tripeptide alone against Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae, with significant reduction in viability obtained via inclusion of 30% w/w ciprofloxacin.Challenges exist to the use of peptides as antimicrobial therapeutics. There is currently only one licensed self-assembled peptide therapeutic; the injectable β-sheet forming octapeptide lanreotide administered for the relief of neuroendocrine symptoms . The licfloxacin . The hydStaphylococcus aureus. [Staphylococcus aureus overnight. The work demonstrated the ability of FmocFF to disassemble and reassemble when added to the flax substrates. Scanning electron microscopy suggested that the higher the concentration of silver present, the more uniform the nature of the flax coverage. Precipitates formed with the 2% w/v gels thus homogeneity was not achieved. Antimicrobial studies demonstrated a reduction in bacterial numbers with an increased silver concentration. Bacterial adhesion and biofilm formation was also investigated. Incorporation of silver was necessary to prevent biofilm formation and the minimum concentration of silver needed to achieve this effect was 0.1% weight.Paladini and co-workers investigated the antimicrobial effects of silver incorporated into ultrashort Fmoc diphenylalanine (FmocFF) hydrogels on  aureus. . HydrogeBacillus subtilis and Gram-negative Escherichia coli for up to 30 days. Stability was also increased compared with silver nanoparticles alone suggesting the peptide hydrogel protects silver nanoparticles from oxidation. The hydrophobic character of ultrashort self-assembling peptides may also allow the incorporation of other highly lipophilic antimicrobial drugs.The amphipathic peptide, FmocFFECG, contains both hydrophobic and hydrophilic moieties. The presence of carboxylic acid functionalities allowed adsorption of silver nanoparticles into the peptide motif . Activit3R6TAT  assembles to form micelles with a hydrophobic cholesterol core and hydrophilic peptide exterior [in vivo in an infected Staphylococcus aureus induced meningitis rabbit model. This work highlights the ability of assembled peptides as tailored molecules to achieve targeted antimicrobial effects in challenging areas of drug delivery.Peptides also provide access to infections in the brain. Research by Liu demonstrated how the peptide CGexterior . This peKey problems associated with peptide design namely, high manufacturing costs and susceptibility to enzymatic degradation, mean there is still a need to develop compounds that have similar properties to conventional antimicrobial peptides in physiological conditions namely: cationic charge, hydrophobicity and amphiphilic nature to enable antimicrobial action and self-assembly. Peptidomimetics and other synthetic oligomers are thus coming to the forefront of current and future research plans. The design of ultrashort peptides has the potential to create functional peptide molecules with a relatively reasonable manufacturing cost. Our own group has moved research forward in this area with the synthesis of a variety of ultrashort and more cost-effective cationic naphthalene containing peptide hydrogels, which are highly active against biofilm forms of biomaterial related pathogens . The ver


De novo assembled transcriptomes, in combination with RNA-Seq, are powerful tools to explore gene sequence and expression level in organisms without reference genomes. Investigators must first choose which high throughput sequencing platforms will provide data most suitable for their experimental goals. In this study, we explore the utility of 454 and Illumina sequences for de novo transcriptome assembly and downstream RNA-Seq applications in a reproductive gland from a non-model bird species, the Japanese quail (Coturnix japonica). Four transcriptomes composed of either pure 454 or Illumina reads or mixtures of read types were assembled and evaluated for the same cost. Illumina assemblies performed best for de novo transcriptome characterization in terms of contig length, transcriptome coverage, and complete assembly of gene transcripts. Improvements over the Hybrid assembly were marginal, with the exception that the addition of 454 data significantly increased the number of genes annotated. The Illumina assembly provided the best reference to align an independent set of RNA-Seq data as ∼84% of reads mapped to single genes in the transcriptome. Contigs constructed solely from 454 data may impose problems for RNA-Seq as our 454 transcriptome revealed a high number of indels and many ambiguously mapped reads. Correcting the 454 transcriptome with Illumina reads was an effective strategy to deal with indel and frameshift errors inherent to the 454 transcriptome, but at the cost of transcriptome coverage. In the absence of a reference genome, we find that Illumina reads alone produced a high quality transcriptome appropriate for RNA-Seq gene expression analyses. Until recently, evolutionary and population-genomic research was restricted to the small number of taxa considered model organisms. Modern next-generation sequencing technologies offer the opportunity to generate massive (and increasing) amounts of sequence data easily and affordably. Today, the potential for large-scale genomic investigations exists for virtually any study system de novo transcriptome assembly de novo transcriptomes directly from Illumina data are emerging de novo assembly with each data type separately and in combination, and compare the various transcriptomes in terms of quality and utility for RNA-Seq. Our objective was to model approaches taken by those studying genomics of non-model organisms, considering cost as a potential limiting factor. Thus, we sequenced our transcriptome with both 454 and Illumina technologies at depths that were approximately the same cost  sequencing platform to facilitate  ∼$5000, .Coturnix japonica) possess a well-developed foam gland that produces a viscous secretion that is whipped into a stiff foam by contractions of the cloacal sphincter muscle Our transcriptome data derive from a reproductive tissue of a non-model species. Male Japanese quail , misassembled paralogs, chimeras, and/or partial transcripts Coturnix quail do not have a well-annotated genome available, Japanese quail diverged ∼34 million years ago from the chicken  and exhibit conserved synteny and chromosomal structure with the chicken genome We initially evaluated the transcriptome assemblies with standard metrics based on transcript length . 2, we immediately dissected out foam glands and froze samples on liquid nitrogen. We extracted RNA with the Agencourt RNAdvance Tissue Kit (Beckman Coulter) following the manufacturer's instructions with the exception that we performed half-reactions. RNA quality and concentration was assessed by agarose gel electrophoresis and NanoDrop spectrophotometry. We checked for RNA purity and integrity using an Agilent 2100 BioAnalyzer.Japanese quail were lab-reared and housed on a 16∶8 light:dark cycle. All study males were sexually mature, had phenotypically normal foam glands, and produced normal foam complements. A foam gland from a Japanese quail male approximately one year old was used to generate the 454 data. Foam glands from six Japanese quail males (two were one-year old and four were five months old), were used to generate the Illumina data for transcriptome assembly. For the independent RNA-Seq assessment, we sampled foam glands from six different Japanese quail males on winter light conditions  with testosterone replacement. Testosterone-replaced males have phenotypically normal foam glands and produce foam We isolated mRNA from one µg total RNA, synthesized first-strand cDNA and generated ds cDNA following the manufacturer's instructions for the SMART Polymerase Chain Reaction (PCR) cDNA Synthesis Kit , with the exception that we used SuperScript III Reverse Transcriptase (Invitrogen) as the reverse transcriptase and made adjustments accordingly. We amplified the cDNA, confirmed successful amplification via agarose gel electrophoresis, and cleaned the PCR products with the QIAquick PCR Purification Kit (Qiagen). We partially normalized our library subjecting amplified cDNA to hybridization and double-stranded nuclease (DSN) digestion following instructions from the TRIMMER cDNA Normalization Kit (Evrogen) except using only 1/8 and 1/16 concentrations of DSN. Size selection was performed with the QIAquick Gel Extraction Kit (Qiagen) according to manufacturer's instructions. We enzymatically fragmented the dsDNA with NEBNext dsDNA Fragmentase , end polished using T4 polymerase , phosphorylated 5′ ends with T4 kinase , added an adenine to 3′ ends with NEB Taq , and ligated Multiplex Identifier (MID) Adaptor #1 for GS FLX Titanium chemistry (Roche/454 Life Sciences) to ds cDNA using T4 ligase . Throughout the normalization, end polishing, and ligation procedure, the ds cDNA was cleaned with the QIAquick PCR Purification Kit (Qiagen) when necessary. Cornell University's Genomics Facility at the Institute of Biotechnology performed ½ plate of 454 GS FLX sequencing with Titanium chemistry on the resulting library (Roche/454 Life Sciences) in April 2010.In January 2012, six Illumina libraries for the transcriptome assembly were prepared from approximately 1.2 µg total RNA using the TruSeq RNA Sample Preparation Kit (Illumina) following the manufacturer's instructions. We also prepared six samples from testosterone-replaced males for the independent RNA-Seq evaluations. All twelve samples were tagged with a unique adapter index, pooled, and single-end sequenced on one lane of an Illumina HiSeq 2000, with a target read length of 100 bp. Sequencing was performed by Cornell University's Genomics Facility at the Institute of Biotechnology in April 2012. Raw data for the sequencing runs is reported in http://sourceforge.net/projects/seqclean/) was used to trim low complexity sequences and short sequences (<90 bp). MID-1 and SMART adaptors were trimmed using both SeqClean and NextGENE (Softgenetics). We assembled the reads into unigenes using two rounds of iAssembler Initial quality filtering of reads was performed by the Cornell University's Genomics Facility at the Institute of Biotechnology. SeqClean (http://code.google.com/p/ea-utils/wiki/FastqMcf) to remove Illumina adaptors, trim low-quality terminal ends, discard short sequences, and filter reads. Fastq-mcf scans a sequence file for adapters and, based on a log-scaled threshold, determines a set of clipping parameters by initially evaluating a subsampled portion of the data. We used fastq-mcf with default parameters, except that we subsampled one million reads for threshold estimation, quality filtered for mean phred scores <20, and set the percentage of bad reads causing cycle removal to 1. We merged the six libraries into a single file and assembled a transcriptome using Trinity release 2012-06-08 with default parameters Initial quality filtering and barcode removal were performed by Cornell University's Genomics Facility at the Institute of Biotechnology. We used fastq-mcf version 1.04.636 . Nesoni utilizes the SHRiMP short read mapper to align short reads to an assigned reference We used Illumina data to correct errors with a 454 transcriptome using the Nesoni pipeline, version 0.85  via the BioMart tool For each assembly, we calculated standard metrics of quality including number of contigs, average contig length, median contig length, N50 (median contig size weighted by length), the distribution of contig lengths, and summed contig length G. gallus assembly: WASHUC2) were downloaded via the BioMart tool i.e., duplicates, alternative splice variants) by self-BLAST following Hornett and Wheat −6, >90% similarity, and >33 amino acids in length, we removed the shorter of the two proteins. All BLAST steps were performed in parallel via Cornell University's Computational Biology Application Suite for High Performance Computing. The reciprocal best blast method was used to determine orthologs with a cutoff e-value of 1 * 10−6We used data from the chicken to identify orthologs. All chicken protein sequences from Ensembl version 68  was used to generate statistics about the quality of the alignments to the various assemblies including the number of mapped/unmapped reads and the number of indels per 100,000 bp identified between each assembly and the majority of the RNA-Seq data. We calculated the number of uniquely mapped reads with samtools We produced an independent Illumina RNA-Seq dataset from foam glands of six different foam-producing Japanese quail males to evaluate the utility of our various assemblies for gene expression analyses. The RNA-Seq data were merged into a single file and aligned using the Burrow-Wheeler transform as implemented in the aln algorithm of BWA with default parameters except that -q was set to 20 Raw data have been deposited on the Short Read Archive under accession numbers SRR1346108 and SRR1352724.All animal procedures were approved by Cornell University's Institutional Animal Care and Use Committee under permit 2002–0117.We sequenced foam glands with the 454 and Illumina platforms and assembled the raw data into four transcriptomes, each having approximately the same sequencing cost: two made solely from each type of data , one that used Illumina data to correct errors in the 454 transcriptome (Corrected 454), and one that used both kinds of data as input (Hybrid). High quality assemblies possess near full-length contigs representing most of the actual transcriptome. We first evaluated each transcriptome assembly and the Chicken coding sequence set using a suite of standard metrics The Illumina assembly displayed the highest values across most standard metrics of transcriptome quality, followed by the Hybrid assembly . The disde novo assembled transcriptomes produced an excess of very short contigs (∼200 bp) relative to the Chicken coding sequences, but the Hybrid and Illumina assemblies had many more long contigs  can produce frameshifts, in which case downstream analyses reliant on properly called open reading frames would be difficult. Frameshifts can create premature stop codons, resulting in shorter open reading frames. We predicted open reading frames g frames . All of g frames . The 454g frames . Only thg frames ; Table 2de novo transcriptome assembly have found that contig length or gene recovery (but not accuracy) are negatively influenced by increased polymorphism Previously, approaches combining both 454 and Illumina data revealed significant improvements over either single technology using similar metrics De novo assembled transcriptomes from non-model species rely on BLAST-based annotations to provide information about gene identity and function. We exploited the fact that quail and chicken are closely related and determined quail-chicken orthologs via reciprocal best BLAST If a research goal is to annotate the maximum number of genes, combining annotations from hybrid and single-data assemblies is the preferred method . The 454Contigs from optimal assemblies represent full, not partial, gene sequences. We assessed how well contigs from each assembly reproduced ortholog length by calculating the “ortholog hit ratio” . This rade novo assembled transcriptomes  outperforms mapping to ence set . Thus, wet al.in silico to the zebra finch transcriptome and mapped simulated RNA-Seq reads, also from zebra finch, back to the various transcriptomes. Their datasets accounted for simple differences due to nucleotide polymorphisms and indels, but did not incorporate more complex forms of variation that could affect the ability to map RNA-Seq data . As we utilized non-simulated data, our reference-based mapping approach encompassed both simple and complex forms of sequence divergence that occurred after the Japanese quail and chicken lineages split. Increasing transcriptome complexity  negatively affects both de novo transcriptome assembly and the ability to quantify gene expression Differences in the nature of the data examined may explain the disparity between our results and previous work. To mimic reference-based mapping, Vijay de novo transcriptome assembly and utility for RNA-Seq analyses in a non-model species. The Illumina assembly often performed the absolute best in standard assays of transcriptome quality, though both the Hybrid and Illumina assemblies produced longer contigs covering more of the transcriptome than 454-based assemblies. Hybrid and Illumina assemblies also afforded more gene annotations that better reproduced ortholog lengths. However, if a goal is to identify the maximum number of annotations, utilizing both 454 and Illumina is preferred, as each contributes a significant number of annotations. Correcting the 454 library with Illumina data drastically reduced the error rate in terms of indels and premature stop codons, but at the cost of contig length and gene annotation. The Illumina assembly offered the best reference for RNA-Seq data, delivering the highest number of uniquely mapped reads by far. Our results may be unsurprising given the vast differences in the number of reads generated by the two technologies. However, cost is often a limiting factor when working with non-model species and we spent approximately the same amount of money to generate both types of data.We compared assemblies generated from mixtures of 454 and Illumina reads for De novo assemblies made from paired-end Illumina sequences are likely to be even better than the results obtained here. Moreover, our Illumina data averaged 20 million reads per sample, which is well within the range the suggested number for robust detection of differential gene expression  that later served as a reference for mapping RNA-Seq reads, generally generated from a separate Illumina run e.g., . From seTable S1Summary statistics of raw data generated for assemblies.(DOCX)Click here for additional data file.


We suggest that reconstruction of ancestral viruses is a useful tool for experimental and computational investigations of virulence and viral evolution. Importantly, ancestral reconstruction can be done even on the basis of a set of sequences that all correspond to non-functional variants.RNA viruses have the highest known mutation rates. Consequently it is likely that a high proportion of individual RNA virus genomes, isolated from an infected host, will contain lethal mutations and be non-functional. This is problematic if the aim is to clone and investigate high-fitness, functional cDNAs and may also pose problems for sequence-based analysis of viral evolution. To address these challenges we have performed a study of the evolution of classical swine fever virus (CSFV) using deep sequencing and analysis of 84 full-length cDNA clones, each representing individual genomes from a moderately virulent isolate. In addition to here being used as a model for RNA viruses generally, CSFV has high socioeconomic importance and remains a threat to animal welfare and pig production. We find that the majority of the investigated genomes are non-functional and only 12% produced infectious RNA transcripts. Full length sequencing of cDNA clones and deep sequencing of the parental population identified substitutions important for the observed phenotypes. The investigated cDNA clones were furthermore used as the basis for inferring the sequence of functional viruses. Since each unique clone must necessarily be the descendant of a functional ancestor, we hypothesized that it should be possible to produce functional clones by reconstructing ancestral sequences. To test this we used phylogenetic methods to infer two ancestral sequences, which were then reconstructed as cDNA clones. Viruses rescued from the reconstructed cDNAs were tested in cell culture and pigs. Both reconstructed ancestral genomes proved functional, and displayed distinct phenotypes  Positive-strand RNA viruses, including classical swine fever virus (CSFV), have high mutation rates compared to eukaryotic organisms due to the lack of proofreading by their RNA-dependent RNA polymerase . This inThe analysis of viral genotype composition has typically relied on clonal approaches and subsequent sequencing of partial genomes. However, Next-Generation Sequencing (NGS) has rapidly become the preferred technology for addressing this issue . Using Nin vitro and in vivo analyses.Here we present a study of the evolution in the CSFV strain “Roesrath”, a recent European genotype 2.3, which displays moderate virulence in domestic pigs and wild boar , 14]. T. T14]. TThe CSFV strain “Roesrath” was used for the experiments . Two different cell culture passages derived from the same isolate were used: the CSFV_Roesrath_P5, which was a fifth passage sample from PK-15 cells whereas the CSFV_Roesrath_P2 was a second passage sample of the same isolate.NotI sites and with a T7 promoter upstream of the cDNA sequence using primers CSFV-Ros_Not1-T7-1-59 and CSFV-Ros_12313aR_Not1 . Individual colonies were propagated on LB plates supplemented with chloramphenicol, and screened following restriction enzyme digestion. PCR products for the in vitro transcription and the sequencing were obtained from each cloned cDNA using same forward primer and CSFV-Ros_12313aR  for detection of viral proteins using microscopy. Cell supernatants from the replication competent transcripts were passaged onto uninfected PK-15 cells and incubated for a further 72 hours.de novo assembler (Roche) and mapped to the CSFV Roesrath reference sequence (GU233734) by the BWA aligner using the BWASW algorithm  on on26] onAncestral reconstruction of the internal nodes was performed using PAML . The BasThe reconstruction of cDNA clones was performed as previously described . BrieflyVirus growth was determined by growth curves as previously described . Briefly5 TCID50/100 μl. Body temperature and clinical signs were observed on a daily basis. Blood samples and oral swabs were taken at days 0, 2, 4, 7, 10, 14, 21, 28 and 42 post-infection. After day 42, the pigs were sacrificed (electro-stunning and subsequent exsanguination) and subjected to post-mortem examination and sampling. The IDEXX CSFV antibody ELISA  and neutralisation tests were performed on the serum samples as previously described , 3, 35]. W. W35]. Win vivo variation found for other genotype 2 medium virulent strains . I. Iin vivThis study combines reverse genetics and NGS for exploring viral population structure in depth. NGS alone is limited to SNV calling and reconstruction of haplotypes, but the latter is mostly useful in highly heterogeneous populations, where overlaps of SNVs between individual sequencing reads are abundant. We have shown that the additional use of full genome sequencing of cDNA clones for reverse genetics can provide powerful new data, which can be used to determine haplotype structure, explore phenotypes, and identify mutations of interest. We suggest that these approaches with benefit could be applied to other RNA viruses, thereby leading to better understanding of the viral population dynamics of these important pathogens.