Dear Readers, Contributors and Reviewers,I would like to take this opportunity to wish all of you an excellent year in 2010. It isalso important to thank all of you for your kindness, support, patience and interest in theJournal of Applied Oral Science (JAOS) in 2009.After the indexation in the two most prestigious databases (PubMed/MEDLINE and ISI Web ofKnowledge), the JAOS was flooded with a huge number of submissions. My small team and Iwere quite happy and honored with such a demonstration of confidence in our work, but wewere not ready to handle that enormous number of submissions. Therefore, I decided totemporarily block our online system for new submissions in order to properly process andanalyze all the submissions. I hope all the contributors understand that this toughdecision was actually a demonstration of respect to their works. Soon the system will bereopened and some changes will be added. The main new requirement will be that in theinitial submission, all the contributors will be asked to provide the JAOS with the namesand e-mail addresses of four potential reviewers since this is, in my opinion, thebottleneck of the reviewing process. This should help to speed up the reviewingprocess.This year the JAOS will have its first impact factor (IF) from the Journal Citation Report(JCR). I am aware we will have a low IF, but this will be instrumental to guide andencourage us to improve it. I count on everyone’s help, support and suggestions to facethis challenge!Finally, I thank all the reviewers and our editorial board, who kindly and blindlyevaluated works for possible publication in the JAOS in 2009. This silent and generous workis the most important and delicate in the reviewing process, and without this precious helpour journal would not have reached the international respect it currently has.http://www.scielo.br/jaos.Please, feel free to contact me through the new e-mail address: editorjaos@fob.usp.br. Wealso invite all readers, contributors and reviewers to visit our free complete onlinecollection and our online submission system at Kind regards,


Ceramics are very important in the science of dental biomaterials. Among all dental ceramics, zirconia is in evidence as a dental biomaterial and it is the material of choice in contemporary restorative dentistry. Zirconia has been applied as structural material for dental bridges, crowns, inserts, and implants, mostly because of its biocompatibility, high fracture toughness, and radiopacity. However, the clinical success of restorative dentistry has to consider the adhesion to different substrates, which has offered a great challenge to dental zirconia research and development. This study characterizes zirconia as a dental biomaterial, presenting the current consensus and challenges to its dental applications. The most popular dental ceramic systems are silica-, leucite-, lithium disilicate-, alumina-, and zirconia-based materials. Currently, zirconia-based ceramics are the most studied, challenging researches for different reasons.2), also named as “ceramic steel”, has optimum properties for dental use: superior toughness, strength, and fatigue resistance, in addition to excellent wear properties and biocompatibility.Zirconia  is a very strong metal with similar chemical and physical properties to titanium (Ti). Incidentally, Zr and Ti are two metals commonly used in implant dentistry, mostly because they do not inhibit the bone forming cells (osteoblasts), which are essential for osseointegration . 2O3) tetragonal zirconia polycrystal (Y-TZP). Yttria is added to stabilize the crystal structure transformation during firing at an elevated temperature and improve the physical properties of zirconia. Upon heating, the monoclinic phase of zirconia starts transforming to the tetragonal phase at 1187 °C, peaks at 1197 °C, and finishes at 1206 °C. On cooling, the transformation from the tetragonal to the monoclinic phase starts at 1052 °C, peaks at 1048 °C, and finishes at 1020 °C, exhibiting a hysteresis behavior. The zirconia tetragonal-to-monoclinic phase transformation is known to be a martensitic transformation [2), yttria (Y2O3), alumina (Al2O3), magnesia (MgO) and calcia (CaO) have been used as stabilizing oxides. So, as the monoclinic phase does not form under normal cooling conditions, the cubic and tetragonal phases are retained, and crack formation, due to phase transformation, is avoided [Dental zirconia is, most often, a modified yttria YO3 tetragZirconia structures used for dental purposes are fabricated using CAD-CAM (computer-aided design and computer-aided manufacturing) technology in two possible ways. One method mills the fully sintered block of zirconia with no distortion (shrinkage) to the final structure. The disadvantages are the great wear of the grinding tools (burs) and the population of flaws produced during the machining that may lower the mechanical reliability of the structure ,5. In thTraditionally, zirconia is dull white in color and its opacity can mask the underneath structure. Most dental zirconia systems indicate structural dyeing (coloring) to enhance the esthetic . CurrentEven so, the most popular zirconia-based restorations have a zirconia infrastructure that is porcelain veneered to adequate anatomic contour and esthetic. There are two main ways of veneering zirconia infrastructures: the traditional layering technique and the hot pressing method ,7. Both Nonetheless, one of the most important reasons for introducing monolithic zirconia restorations is the significant rate of porcelain fracture from porcelain veneered zirconia-based restorations (6%–25% after three years), which is greater than the fracture rate reported for porcelain fused-to-metal (PFM) systems ,27,28,29The mismatch in some mechanical and thermal properties such as fracture toughness, flexural strength, coefficient of thermal expansion, and elastic modulus affect the bonding between porcelain and zirconia ,36,37,38Tg from 480 °C to 610 °C) [Significant differences on the coefficient of thermal expansion between the zirconia and porcelain influence in the residual stress distribution during the cooling process affecting the reliability of zirconia-based restorations ,38,39,40 610 °C) ,7,40. Du 610 °C) ,38,39. The initiation and propagation of delamination was also reported to be related to the misfit in elastic moduli and fracture toughness of porcelain and zirconia . Pressable veneering porcelains were thought to improve bonding between porcelain and zirconia, but studies are controversial on this matter and most of them showed no significant difference between the traditional and pressing methods ,42. New ., composites and metals), working as an integrated system where diverse stresses, from chewing to para-functional habits , are distributed throughout the system due to appropriate bonding [At first, one could imagine that an all-ceramic restoration would not withstand the intra-oral service. It could be true if the restoration would not be bonded to the tooth structure or remaining restorative materials  and resin-based cements are the primary choices for bonding ceramic restorations to the remaining tooth structure. GIC and resin-modified GIC (RMGIC) are often used to cement acid-resistant ceramics, mostly because these cements are very easy to use. However, the most popular and effective cements for all types of ceramic restorations are the resin-based composites, including the systems containing the 10-methacryloyloxydecyl-dihydrogen-phosphate (MDP) monomer ,45,47.i.e., low bond strength to other substrates [It has been reported that the clinical success of resin bonding procedures for cementing ceramic restorations and repairing fractured ceramic restorations depends on the quality and durability of the bond. The former depends upon the bonding mechanisms that are controlled in part by the surface treatment that promotes micromechanical and/or chemical bond to the substrate ,56,57,58bstrates .Ra) values and that silica coated surfaces showed a significant increase (76%) in the concentration of silicon, which should enhance bonding to resin via silane coupling agents [As zirconia is an acid-resistance ceramic, other methods to produce micromechanical retention have been used, including airborne particle abrasion (APA) systems, often called sandblasting, and coarse diamond rotary instruments. Several studies ,51,58,59g agents ,51,59. Tg agents ,45.2 or N/m) is referred to as the surface energy (γ), or surface tension for liquids  [Therefore, the adhesion between dental ceramics and resin-based composites is the result of a physico-chemical interaction across the interface between the resin (adhesive) and the ceramic (substrate). The physical contribution to the adhesion process is dependent on the surface treatment and topography of the substrate and can be characterized by its surface energy. Alteration of the surface topography results in changes on the surface area and on the wettability of the substrate, which are related to the surface energy and the adhesive potential. In addition, the surface energy of a solid surface is greater than that of its interior where the interatomic distances are equal, and the energy is minimal. In fact, at the surface of the lattice, the energy is greater because the outermost atoms are not equally attracted in all directions. This increase in energy per unit area of surface J/m or N/m i0 mJ/m2) . Therefo0 mJ/m2) .in vivo. Clinical trials on veneered zirconia-based restorations have showed survival rates of 75%–100% [Nevertheless, a clean (no contaminants) and dry surface ensures that the adhesive has the best possible chance of creating a proper bond with the adherend. In addition, the wettability of the adherend by the adhesive, the viscosity of the adhesive, and the morphology of adherend surface influence the ability of the adhesive to make intimate contact with the adherend. Thus, to succeed the challenge of resin-bonding to zirconia-based ceramics, one must consider all aspects listed above ,45. This75%–100% ,29,60,61in vitro studies  fracture initiated in the connector area of fixed partial dentures (FPDs), either at the core-veneer interface or at the gingival embrasure; and 2) chipping of the porcelain veneer  chipping,106,107.The mode of failure is an important aspect of bond strength tests, but it is not commonly reported. A detailed inspection of the fractured surfaces can indicate the failure mode of a bonded assembly. The fracture behavior of adhesive interfaces depends on the stress level, the flaw distribution, material properties, and environmental effects. Therefore, fracture surface characterization combined with analyses of fracture mechanics parameters are of great importance to understand and predict bonded interface reliability and also to reduce the risk for data misinterpretation such as the inference that the bond strength exceed the cohesive strength of the substrate when the fracture initiates away from the interface ,47,51,55The above rationale on adhesion to zirconia should develop the fundamental basis to understand the clinical performance of bonded zirconia-based restorations, the possible failure causes, and the principles to improve the adhesion mechanisms of resin-based composite bonded to zirconia.


Manihot esculenta Crantz.) varieties developed by the Crops Research Institute of Ghana (CRIG) was studied in paracetamol tablet formulations. The yield of the starches ranged from 8.0 to 26.7%. The starches were basic (pH: 8.1–9.9), with satisfactory moisture content (≤15%), swelling capacity (≥20%), ash values (<1%), flow properties, and negligible toxic metal ion content, and compatible with the drug. The tensile strength (Ts), crushing strength (Cs), and friability (Ft) of tablets containing 5–10% w/w of the cassava starches were similar (p > 0.05) to those containing maize starch BP. The disintegration times of the tablets decreased with increase in concentration of the cassava starches. The tablets passed the disintegration test (DT ≤ 15 min) and exhibited faster disintegration times (p > 0.05) than those containing maize starch BP. The disintegration efficiency ratio (DER) and the disintegration parameter DERc of the tablets showed that cassava starches V20, V40, and V50 had better disintegrant activity than maize starch BP. The tablets passed the dissolution test for immediate release tablets (≥70% release in 45 min) with dissolution rates similar to those containing maize starch BP. The disintegrant potential of native starches of five new cassava ( Starch and its derivatives  are principally used as disintegrants in pharmaceutical tablet formulations. Starch and its derivatives are also used as diluents, binding agents, glidants, and thickeners. Disintegrants are pharmaceutical excipients that are included in tablet formulations with the aim of facilitating the break-up of the compressed tablets into small fragments in aqueous media. The enhanced splitting of the tablets in aqueous media enhances the dissolution, absorption, and bioavailability of orally administered drugs. Other substances employed as disintegrants in pharmaceutical formulations are cellulose and its derivatives , resin and its derivatives, and crospovidone .Native starches derived from botanical sources are commonly employed as disintegrants in pharmaceutical tablet formulations usually in a concentration range of 2–10% w/w . Other fVarious mechanisms of disintegration have been proposed for disintegrants. These mechanisms which have variable application to different types of disintegrants are swelling, capillary action or wicking, strain recovery, heat of interaction, and interruption of particle-particle bonds , 5. SwelThe first disintegrating agents to be used in tablet formulations were native starches of maize, potato, and wheat . In geneThe Crop Research Institute of Ghana (CRIG), Fumesua, is using genetic techniques to develop new cassava varieties with high starch, fiber, nutrient content, and other functional properties. The aim of the current study was to evaluate the disintegrant properties of native starches of five improved cassava varieties developed by CRIG, Fumesua, in paracetamol tablet formulations. Paracetamol was used as a model drug because it has poor flow and compression properties and high capping and lamination tendencies and lacks any inherent disintegrant activity. The effects of the cassava starch disintegrants on the physicomechanical and drug release properties of the formulated paracetamol tablets were evaluated.Manihot esculenta Crantz.), namely, Sika Bankye, Ampong, AW/10/008, 12/0245, and 12/0197 , were obtained from CRIG, Fumesua, Ghana. The cassava varieties were planted in March 2014 and harvested in October 2015, and starch extractions were undertaken within two days of harvesting. Maize starch BP  was used as the reference disintegrant (coded V60). Paracetamol BP , magnesium stearate , talc and lactose , polyvinylpyrrolidone (PVP) , sulphuric acid, nitric acid, hydrochloric acid and sodium hydroxide pellets , ethanol 96% , and potassium dihydrogen orthophosphate  were used.Five varieties of matured fresh root tubers of cassava . The total ash, acid insoluble ash, and water soluble ash of the cassava starches were determined using official methods .Vd). The starch powder was dispersed in 70 ml of distilled water and made up to 100 ml with water. After 18 hours of standing the volume of the sediment (Vw) was determined. The swelling index (%) was calculated using the equation: The tapped volume occupied by 10 g of the powdered cassava starch in a 100 ml measuring cylinder was recorded  at 25°C. Five hundred milligrams of the starch powder was added to 50 ml of solvent and allowed to stand overnight. Twenty-five milliliters of the supernatant was placed in preweighed Petri dishes and evaporated to dryness over a water bath and further dried to constant weight in an oven at 100°C. The mass of the residues was determined with an analytical balance  and expressed as the percent solubility of the cassava starch in the respective solvents.M1) of cassava starch powder was weighed into a 100 ml measuring cylinder and the volume occupied (V1) noted. The measuring cylinder was then tapped on a hard surface to consolidate the powder to a constant volume (V2). The fluff density, tapped density, Hausner ratio, and Carr's index were calculated as follows: Thirty grams (h) and the mean diameter (D) of the base of the starch powder cone were determined. The angle of repose (θ) was calculated using the equation A funnel was clamped with its tip 2 cm above a hard horizontal surface. The starch powder was allowed to flow through the funnel until the apex of the powder formed just touched the funnel's tip. The height , arsenic (As), lead (Pb), and mercury (Hg) in the cassava starches was determined with an atomic absorption spectrophotometer (Buck Scientific Model 210V GP) as previously reported , 12.−1 using the FTIR spectrometer by placing the sample into a diffuse reflectance sampler. The spectra of the three samples were superimposed to assess whether or not the principal absorption bands present in the drug and starches are present in the physical mixtures.A Fourier transform infrared spectrometer (FTIR)  was used to determine the compatibility between paracetamol and the cassava starches. The spectra of the individual starches, paracetamol, and the physical mixtures of the drug and starch were recorded by scanning in the wavelength region 4000–400 cmμm) and dried in a hot air oven at 50°C for 2 h. The dry granules were manually screened using a number 16 mesh sieve (1190 μm) and mixed with talc for 5 min in the V-blender. Finally, magnesium stearate was added and blended for a further 5 min and discharged. The different batches of the paracetamol granules in the size range of 595–1190 μm were compressed into tablets (~600 mg) at pressures of 45–50 KN using a Cadmach CTX 26 tableting machine . The tablets were stored for 24 h after compression to allow for elastic recovery and hardening before evaluation.Paracetamol granules (420 g) comprising of paracetamol (83.3%) as active pharmaceutical ingredient, lactose  as diluent, polyvinylpyrrolidone, PVP (4.5%), as binder, talc (1.2%) as glidant, magnesium stearate (0.2%) as lubricant, and cassava starch or maize starch BP  as disintegrant were prepared by the wet granulation method. The required amount of paracetamol was dry-mixed with lactose and starch and moistened with PVP solution and massed for 10 min in a V-blender . The damp mass was manually screened through a number 12 mesh sieve .λ-max of 275 nm using the regression data of the calibration curve  of paracetamol (0.25–1.5 mg/100 ml) in 0.1 M sodium hydroxide.The paracetamol content of the tablets produced was determined using the procedure described in the British Pharmacopoeia . The amoCs) of ten randomly selected tablets from each batch was determined with an Electrolab hardness tester  as described in the British Pharmacopoeia [Ts) was determined using the equation D is the diameter and t is the thickness of the tablet [The crushing strength . Ten tablets weighing ~6.0 g from each batch were placed in the friabilator and it was operated for 4 min at 100 rpm. The tablets were dedusted and reweighed and the difference in tablet weight was determined. The friability was calculated as follows: The disintegration time of the tablets of each batch was determined using an Erweka disintegration apparatus  as described in the British Pharmacopoeia .Cs, Ft, and DT are the crushing strength, friability, and disintegration time, respectively. The disintegration parameter DERc was determined using the formula test is the DER of tablets containing the test disintegrant , while DERreference is the DER of tablets containing the reference disintegrant (V60). When DERc > 1, the DER of the test cassava starch disintegrant is considered to possess better disintegrant action than the reference disintegrant and vice versa [The disintegration efficiency ratio (DER) was determined using the relationship ce versa , 15.y = 624.16x + 0.0283, R2 = 0.9975). Triplicate determinations were performed for each batch. Graphs of percentage drug released were plotted against time to establish the dissolution profiles of paracetamol.In vitro tablet dissolution tests were undertaken with an Erweka dissolution apparatus  under sink conditions. Dissolution was determined in 900 ml phosphate buffer pH 5.8 at a paddle speed of 50 rpm and temperature of 37 ± 0.5°C. A tablet was carefully placed into each vessel to exclude air bubbles from its surface. At times 5, 10, 15, 30, 45, and 60 min, 20 ml of the dissolution medium was withdrawn and filtered and replaced with an equal volume of medium. The filtrates were diluted to 50 ml with 0.1 M NaOH and the absorbance of each solution was measured at 257 nm with a UV spectrophotometer . The amount of paracetamol dissolved was determined using the regression data equation  followed by Tukey's post hoc multiple comparison test with GraphPad Prism version 5.00 for Windows . At 95% confidence interval, Manihot esculenta Crantz.) after extraction produced starch yields ranging from 8.0% to 26.8% with the ranking of V20 > V40 > V10 > V30 > V50. Cassava starch yield was affected by the cassava variety used. Other factors which are known to affect cassava starch yield include processing factors [All the five varieties of cassava  and 1561.11 cm−1, 1504.98 cm−1 (amide II band) which are characteristic of the drug. The cassava starches showed intense functional groups at 3274.71 cm−1 (free hydroxyl group) and 1336.55 cm−1 and 997.37 cm−1  which characterized them as starches. The physical mixtures contained all the principal bands of the two constituents and showed no shifting of the peaks, indicating the stability and compatibility of the starches with the drug.n = 20) [p > 0.05) between the crushing strength of tablets containing maize starch and the cassava starches.Paracetamol tablets containing different concentrations of the cassava starches as disintegrants were produced by the wet granulation technique. The starch disintegrants were added to the tablet formulations intragranularly. n = 20) , except n = 20) , and alln = 20) . There wp > 0.05) between the tensile strength of tablets containing the cassava starches and that of maize starch BP, even though the tensile strength of the latter was higher. Friability test is used to evaluate the capacity of tablets to withstand abrasion and chipping associated with handling, packaging, and shipping. Apart from tablets which contained 10% w/w of cassava starch V10, all the tablets passed the friability test (tablet loss ≤ 1%). An increase in cassava starch disintegrant concentration did not produce any specific effect on tablet friability and there were no significant differences (p > 0.05) between the friability of tablets which contained cassava starch and those containing maize starch BP. The disintegration of a tablet is affected by the temperature of the disintegration medium, the nature and amount of binder used, force of compression, and the nature and amount of disintegrant used [p > 0.05) were observed between the disintegration times of tablets containing cassava starches and those containing maize starch BP.Tablets should have strong tensile strength to withstand pressure due to handling, film-coating, and packaging but must be weak enough to allow drug release after administration. In general, the tensile strength of the tablets reduced with increase in cassava starch concentration. There were no significant differences (ant used . An incrant used . No signCt/Ft) is a better measure of the mechanical strength of tablets than crushing strength as it removes the weakness related to the friability of a tablet [Ct/Ft values have strong mechanical strength [c values of tablets containing the cassava starches V40, V50, and V20, which were generally >1. This provides an indication that these experimental or test cassava starches had better DER values than that of the reference disintegrant. On the other hand, tablets containing cassava starches V10 and V30 generally had DERc values <1 which shows that the two cassava starches had inferior DER values than that of the reference disintegrant.The crushing strength-friability ratio  and crushing strength (Cs) of the tablets decreased with increase in starch concentration. All tablets containing the cassava starches disintegrated in aqueous medium in less than 15 minutes and the disintegration time decreased with increase in starch concentration. The friability of the tablets was generally unaffected by an increase in starch concentration. The disintegration efficiency ratio (DER) of tablets containing the cassava starches generally increased with increase in starch concentration. Tablets containing different concentrations of the cassava starches released more than 70% of the drug in phosphate buffer pH 5.8 at 45 minutes and were comparable to tablets containing maize starch BP. Even though starch from cassava variety V40 had a rather low starch yield of 11.2%, it appeared to be the preferred cassava starch disintegrant as the different concentrations of the starch generally produced tablets with superior physicomechanical and in vitro drug dissolution characteristics.The results showed that the five cassava varieties have low starch yields. The starches contained negligible amounts of toxic metal ions and generally exhibited good physicochemical properties required for use as pharmaceutical excipients. The five cassava starches employed as intragranular disintegrants in concentrations of 5–10% w/w produced paracetamol tablets with satisfactory physical properties comparable with maize starch BP. There were no interactions between the starches and the active pharmaceutical ingredient and excipients used in producing the tablets. Generally, the tensile strength (


This review highlights the preclinical and clinical research based on the use of nano- and micro-carriers in thrombolytic drug delivery. Ischemic heart and stroke caused by thrombosis are the main causes of death in the world. Because of their inactivation in the blood, high doses of thrombolytics are administered to patients, increasing the risk of intracranial hemorrhage. Preclinical research conducted with lipid, polymer or magnetic nanoparticles loaded with thrombolytic drugs showed an enhancement of thrombolysis and a reduction of undesirable side effects. Targeted nanocarriers exhibited an increased accumulation into clot. Clinical trials were already conducted with lipid-based microbubbles combined with ultrasound and thrombolytic drug and showed thrombolysis improvement. Future validation of nanosystems is awaited in clinic. This research opens new strategies for the management of thrombotic diseases. To dissolve a thrombus, thrombolytic drugs are administered, but they are rapidly inactivated in the blood. High amounts are thus injected to patients with the risk to develop intracranial hemorrhages. Nanocarriers and microbubbles have been tested in preclinical models to deliver thrombolytic drugs. These systems have the advantage to protect the drug from the degradation. In clinical trials, galactose and lipid-based microbubbles associated to ultrasound and thrombolytic drugs showed an enhancement of thrombolysis. Other systems are also expected with new drugs combined or not with endovascular intervention to treat ischemic heart or stroke. Atherosclerosis is a multifactorial and slowly progressing pathophysiological disease. It is responsible for 17.3 million deaths per year. Among these, myocardial infarction and ischemic stroke are, and will remain the principal cause of death in the world [The stages of this disease are now understood in detail. Atherosclerotic lesions begin as fatty streaks in lesion-prone areas in aortic bifurcations. These regions exposed to a disturbed flow may develop an activated endothelium. Low-density lipoproteins (LDLs) derived cholesterol extravasate through the defective endothelium into the subendothelial space. There, LDLs are oxidized (oxLDLs) by enzymes such as myeloperoxidase, 15-lipoxygenase or nitric oxide synthase. The recruitment of monocytes is stimulated in part by oxLDLs and is regulated by adhesion molecules expressed on the surface of endothelial cells . Monocytes are subsequently differentiated into macrophages A. The maAtherosclerosis alone is rarely fatal, but the complications induced by thrombosis, myocardial ischemia and stroke, are the most common causes of death in western societies . ArteriaThrombolytic agents are able to induce thrombus lysis by the degradation of fibrin contained in clots . Clot diThe development of new formulation of these drugs gains more and more interest. A promising strategy consists of the use of nano- and micro-carriers ,15. The in vivo, possess low toxicity, and are easy to biofunctionalize. Depending on their composition, hydrophobic or hydrophilic drugs can be incorporated or attached. The formulations of nano- and micro-carriers include spheres, capsules and vesicles.Several types of materials, polymeric, lipid or metallic based, can be employed to deliver thrombolytic drugs . These mSome factors such as the size, the surface charge and the presence or not of a polymer coating, affect the clearance and the biodistribution of nano- and micro-carriers. The size can be modulated during the synthesis of nanoparticles. The charge and the composition of the surface determine the clearance by the monocyte macrophage system (MPS). Following intravenous administration, the naked nano- and micro-carriers are cleared by the MPS and the recognition is induced by the attachment on the surface of the carriers of plasma proteins named opsonins  . To avoiAmong the lipid-based, only liposomes and some types of microbubbles have been used in thrombo-therapy. Described 50 years ago by Bangham, liposomes represent the most used carriers to deliver active drugs. Liposomes are defined as bilayer phospholipid vesicles, mimicking the cellular membrane, enclosing an aqueous core. Since their discovery the preparation methods were diversified . The choco-glycolic acid) (PLGA), an FDA and EMA-approved polymer, is another promising candidate for drug encapsulation. It is largely used in medical research because of its good biodegradability and biocompatibility.Synthetic and natural polymers are used in the design of nano- and micro-carriers charged with thrombolytic drugs. The polymers have the advantage of being more resistant to mechanical constraints than lipid-based ones. In addition to their biocompatibility and biodegradability, polymer carriers can be tuned in terms of size, porosity and hydrophobicity. Polymers are either functionalized with reactive groups, such as primary amines, for reaction with the drug further to the nanoparticle synthesis or are designed to directly interact with the therapeutic agent during the nanoparticle formulation. The first strategy leads to systems where the drug is charged at the surface of the particle. It has especially been applied to magnetic particles coated with a polymer shell . The mosAnother mechanism of nanoparticle formation is the ionic gelation process. Chitosan nanoparticles are obtained by this method allowing for drug entrapment during the assembly process. Chitosan is a natural water soluble polysaccharide. It possesses cationic and hydrophilic properties as well as good biodegradability and biocompatibility, low toxicity and immunogenicity . In some3O4) or maghemite (γ-Fe2O3) or a mixture of both, surrounded by a shell made of polymers, to improve colloidal stability  treatment, Leach et al. encapsulated the enzyme into naked liposomes and into a water-soluble double emulsion polymer (PEG and PVA). The authors noted some difficulties with the stability of the liposomes. Only 30% of the initial SK was entrapped within liposomes. The SK yield into polymeric porous particles was 82%. When injected into rabbits with autologous carotid artery thrombosis, the time necessary for reperfusion was significantly reduced: on average 7.3 min were necessary for SK loaded into polymers, 19.3 min for SK-liposome and 74.5 min for free SK [In order to reduce side effects induced by SK  peptide in order to target GPIIb/IIIa receptor expressed at the surface of the activated platelets in thrombi  nanoparticles covalently coupled with UK. In order to target femoral thrombi generated in dogs, they coupled antifibrin antibody on the surface of the nanoparticles. When injected into the animals, the thrombus dissolution was higher in animals receiving PFC-UK loaded and antifibrin functionalized nanoparticles than in control animals receiving irrelevant IgG targeted UK PFC nanoparticles [In an interesting approach, Marsh articles . This thet al. have successfully loaded UK and RGD to the surface of a microbubble-based ultrasound contrast agent. They observed an aggregation at the surface of femoral arterial thrombi in rabbits. However, the authors did not quantify the in vivo thrombolytic activity [Mu activity . In rabbactivity .tPA is encoded in humans by a gene on chromosome 8 and is produced by endothelial cells. tPA has two inhibitors, PAI-1 and PAI-2, which belong to the serpin superfamily. The tPA residues 296–304 are critical for the interaction with PAI-1. It shows a limited half-life of about 4–6 min .et al. have loaded rtPA (Cleactor®) on cationizated gelatin and PEG-grafted nanoparticles. When injected into rabbits with balloon injury of right femoral artery, the half-life of rtPA was three times enhanced due to its complexation with gelatin. In order to induce thrombolysis they applied ultrasound  for up for 60 min [Uesugi r 60 min . A complr 60 min .et al. developed PLGA nanocarriers loaded with rtPA (Alteplase). In order to enhance the accumulation in a rat model of abdominal aortic thrombi induced using ferric chloride, they covered the nanoparticles with a chitosan shell and targeted it with cyclic RGD peptide via a carbodiimide bond. The rtPA loaded into the nanoparticles showed a prolonged half-life. The authors reported some limitations such as the encapsulation efficacy of the rtPA (between 54 and 64%) simultaneously to a partial loss of rtPA activity [Zhou activity .3O4) core that responds to an external magnetic field with superparamagnetic properties. The nanoparticles were concentrated into a thrombus under external magnetic guidance after injection into rats with iliac artery embolisms (Magnetic nanoparticles coated with polyacrylic acid (PAA) were covalently coupled with an amine group of rtPA (Alteplase). Magnetic nanoparticles show the advantage of being made of a magnetite . The nanoparticles were injected in pigs with ferromagnetic stents in their coronary artery. The restoration of blood flow with rtPA loaded nanoparticles was reached with a lower amount of drug compared with free rtPA [These nanocarriers reduce the effective dose of active principle injected but also increase the local concentration and prevent side effects. A limitation of this method could be linked to the fact that the external magnetic field decreases with the tissue depth, and thus makes it difficult to target a site deeper in the body. Kempe ree rtPA .The McCarthy team coupled rtPA (Alteplase) to cross-linked dextran-coated iron oxide nanoparticles (CLIO). In order to increase the distance between the fibrinolytic drug and the particles and to minimize the steric interactions they added a PEG spacer moiety. The nanoparticles were moreover functionalized by covalent grafting of activated factor XIII (FXIIIa) via peptide affinity ligands. They injected these targeted nanoparticles (CLIO-FXIII-PEG-rtPA) into mice with pulmonary embolisms obtained with human clots. This choice was based on the facts that the murine plasminogen system is tenfold less sensitive to human tPA. The authors concluded that the targeted nanoagent displayed similar thrombolytic potential as free tPA . These set al. developed liposomes, either coated with PEG or not, and decorated with a peptide sequence of fibrinogen gamma-chain targeting GPIIb/IIIa. Entrapment efficacy of rtPA (Alteplase) varied from 12 to 26% for non-PEGylated liposomes and from 36 to 52% for PEGylated liposomes. These nanoparticles were injected into rats with inferior vena cava thrombosis induced with a FeCl3 solution. An enhancement of 35% of thrombolytic activity was observed for rtPA-loaded into targeted liposomes when compared with native rtPA. Tested ex vivo on human clot, liposomal rtPA showed a slightly lower activity compared with native rtPA, this being probably linked to the incomplete release of the drug from liposomes [The loading of rtPA (Alteplase) into liposomes obtained using the freeze-thawing preparation method, did not alter the fibrinolytic activity of the drug. The rtPA loaded into PEGylated liposomes showed 21-fold prolonged circulation time compared with free rtPA . Absar eiposomes .et al. developed ELIP loaded with rtPA (Alteplase) in the core (15%) or associated with the phospholipid bilayer (35%). The rtPA solution was loaded into liposomes during the hydratation of lipid film. ELIP loaded with rtPA were injected into rabbits with abdominal aortic thrombi exposed to ultrasound (pulsed ultrasound 5.7 MHz for 2 min). The degree of recanalization determined by Doppler flow measurements, showed that ELIP charged with rtPA had similar efficacy to free rtPA for thrombus dissolution in vivo. Injection of saline (control), empty ELIP, or empty ELIP associated with ultrasound did not show any thrombolytic effect [An interesting approach to enhance the thrombolysis is the use of ultrasound as adjuvant therapy. This strategy was developed with echogenic liposomes as well as with microbubbles. Echogenic liposomes (ELIP) are composed of a phospholipid bilayer enclosing both gas and liquid. The advantage of this system is the follow-up by echography of thrombus evolution before, during and after thrombolysis. Moreover, ELIP are not only ultrasound contrast agents but also potential vectors for thrombolysis. Laing c effect . The recc effect . This enc effect .et al. developed perfluorocarbon based echogenic liposomes targeted with a RGD peptide loaded or not with rt-PA (Monteplase). They injected them intravenously into rabbits with thrombus in ilio-femoral arteries and applied ultrasound. A higher recanalization rate (nine out of ten rabbits) was observed when ultrasounds were applied, compared with that of animals receiving nontargeted liposomes (two out of ten rabbits) or rtPA monotherapy (four out of ten rabbits) [In order to enhance accumulation into thrombus, Hagisawa rabbits) .Microbubbles with a size from 2 to 8 µm represent another class of lipid based carriers used in thrombolytic strategies. They are composed of a shell made of phospholipids, polymers or albumin, and a core of air or high molecular weight gas . The effet al. showed on rats with filament occlusion of the right middle cerebral artery that the association of rtPA (Alteplase) with microbubbles and ultrasounds completely restored the blood flow, while rtPA alone partially improved hemispheric perfusion [Nedelmann erfusion . In a raerfusion . These set al. included 111 patients with middle cerebral artery occlusions. The 38 patients who received galactose-based microbubbles, rtPA and 2 MHz ultrasound pulse showed a better recanalization rate than the other 73 patients who received either rtPA and ultrasound or rtPA only [et al. showed that half of patients receiving rtPA, 2 MHz continuous TCD monitoring and perflutren-lipid based microbubbles demonstrated a complete recanalization rate (six patients out of 12) while none of the patients receiving only rtPA reached a complete recanalization [Microbubbles are the only platform tested in the clinic for thrombolytic therapy. In clinical trials, they were associated or not with ultrasound and showed encouraging results. Molina tPA only ,59. LatetPA only . Alexandlization . Microbulization .et al. in patients with stroke with basilar artery occlusion. All the 20 patients received intravenous rtPA, ultrasound and galactose based microbubbles. At 24 h, only 50% of patients showed a progressive recanalization while the other 50% did not show any recanalization [et al. showed that the recanalization was similar in patients receiving either galactose-based microbubbles or phospholipid-based microbubbles encapsulating sulfur-hexafluoride, and rtPA associated with 2 MHz transcranial Doppler [Another clinical trial was made by Pagola lization . Rubiera Doppler .One possible explanation of thrombolysis enhancement when microbubbles and ultrasound are associated with rtPA, is the mechanical damage induced by the streaming of microbubbles at the surface of the thrombus allowing a higher diffusion of rtPA. This opens new perspectives for clinical thrombolytic therapy with the aim to reduce the dose and, therefore, hemorrhagic side effects.Nano- and micro-carriers are able to replace the systemic therapy of whole body with a local therapy reducing significantly undesirable side effects. Although ischemic heart and stroke caused by thrombosis are the main causes of death in the world, there is no existing nanocarrier loaded with thrombolytic drug in clinic. Currently, only galactose or lipid-based microbubbles associated to ultrasound with or without rtPA systemic injection have been evaluated in clinical trials and showed an enhancement of thrombolytic effect. In other pathologies such as cancer or infectious diseases, different therapeutic drugs loaded to nanocarriers have already reached the market. This gap could be partly explained by the distinctive features of thrombosis and of the thrombolytic drugs.In the case of heart ischemia or stroke it is necessary to act rapidly within the first minutes or hours (<4.30 h) after the appearance of the symptoms. Thrombolytic drugs administered by systemic way are quickly inactivated by inhibitors in blood. Nanocarriers have the advantage to protect the drug, enhancing its half-life in the blood. Encouraging results were thus obtained on preclinical models with lipid or polymer systems loaded with thrombolytic drugs.The drugs loaded into nanocarriers must keep their therapeutic capacities. The therapeutic drugs are either charged on the surface of the nanoparticles, as for magnetic nanoparticles, or loaded inside nanocarriers, which is the case of polymer or lipid based systems. Another critical point is linked to the amount of drug loaded into nanocarriers. The loading capacity must be maximal in order to reduce the injected quantity of carriers. The preclinical results obtained with liposomes showed a low entrapment efficacy, while a better efficacy was usually obtained with polymer based nanoparticles.An interesting aspect linked to the use of nanocarriers loaded with therapeutic drugs is the capacity to achieve an active targeting of the thrombus, improving the therapeutic efficacy. This was for instance possible by using targeting peptides on the surface of nanocarriers. Another modality of targeting tested on preclinical models was based on the use of magnetic nanoparticles. Although encouraging results were obtained in small animals, this method seems difficult to apply in patients because of the deep localization of the thrombus. Maybe, the use of large and ultrastrong magnets would be a possibility to overcome this limitation in the near future.For all systems, a common limitation is linked to the difficulty to obtain a controlled release. The use of ultrasound can partially control the release into thrombus of therapeutics loaded into echogenic liposomes. However, the ultrasound have a limited penetration into the body, and the use of high frequencies could induce blood vessel lesions. Again, improvements of ultrasound equipment may increase its efficacy.Another important aspect is linked to the thrombus itself in preclinical studies. Some preclinical models are made on animals with an autologous thrombus. However, the affinity of thrombolytics used in clinics for the animal clot is not similar to the affinity for the human one. The use of animal models obtained with a human clot would thus be more relevant. The aging of the used thrombus is also relevant.In conclusion, more in depth preclinical research and developments are necessary to improve the targeted delivery of thrombolytic drugs before their translation into clinical practice.Nanomedicine offers an opportunity and a challenge in the management of a targeted thrombolytic therapy. There are some specific features to take into account for thrombus therapy using nanocarriers. The nanocarrier should load a maximum amount of drug while keeping the thrombolytic efficacy, protect the drug from enzymatic degradation and ensure a rapid release in the thrombus. A balance between all these aspects is mandatory for a better patient care. The development of new materials for nanocarriers and the discovery of new thrombolytic drugs are also considered in the development of nanothrombolysis.Heart ischemia and stroke are the main causes of death in the world.Tissue plasminogen activator but also urokinase and streptokinase are the thrombolytic drugs used in clinic. These drugs are inactivated by circulating inhibitors. Therefore, high amounts are injected to patients in order to obtain a therapeutic effect but with a risk of undesirable side effects such as intracranial hemorrhages.Polymer-, lipid- or magnetic-based nano- and micro-carriers are able to deliver thrombolytic drugs according to their size and structure.In preclinical studies, nanoparticles loaded with either urokinase, streptokinase or recombinant tissue plasminogen activator (rtPA) have been evaluated. As compared with free drug, a lower amount of drug loaded into nanoparticles was necessary to induce a thrombolytic effect. In parallel, a reduction of intracranial hemorrhages was observed.In clinical trials, only galactose and lipid-based microbubbles were evaluated. They were associated to ultrasound and/or rtPA. Enhancement of thrombolytic efficacy was observed with microbubbles associated to ultrasound and rtPA. However, further validation is necessary before a daily clinical use.In conclusion, loading thrombolytic drugs into nanocarriers opens new perspectives for thrombotic diseases therapy.


Staphylococcus aureus manganese transporter protein MntC is under investigation as a component of a prophylactic S.aureus vaccine. Passive immunization with monoclonal antibodies mAB 305-78-7 and mAB 305-101-8 produced using MntC was shown to significantly reduce S. aureus burden in an infant rat model of infection. Earlier interference mapping suggested that a total of 23 monoclonal antibodies generated against MntC could be subdivided into three interference groups, representing three independent immunogenic regions. In the current work binding epitopes for selected representatives of each of these interference groups  were mapped using Hydrogen-Deuterium Exchange Mass Spectrometry (DXMS). All of the identified epitopes are discontinuous, with binding surface formed by structural elements that are separated within the primary sequence of the protein but adjacent in the context of the three-dimensional structure. The approach was validated by co-crystallizing the Fab fragment of one of the antibodies (mAB 305-78-7) with MntC and solving the three-dimensional structure of the complex. X-ray results themselves and localization of the mAB 305-78-7 epitope were further validated using antibody binding experiments with MntC variants containing substitutions of key amino acid residues. These results provided insight into the antigenic properties of MntC and how these properties may play a role in protecting the hostagainst S. aureus infection by preventing the capture and transport of Mn2+, a key element that the pathogen uses to evade host immunity.The  Staphylococcus aureus protein MntC is a metal-binding protein of the ABC-type transporter involved in the acquisition of an essential nutrient, Mn2+, by the pathogen. An earlier study demonstrated that use of MntC as an antigen in experimental vaccine can provide protection against staphylococcal infections in animals and identified three groups of protective monoclonal antibodies induced by the protein. In the current work we employed Deuterium-Hydrogen Exchange Mass Spectrometry (DXMS) to determine binding sites of selected representatives from each of those three groups. DXMS results were further validated using X-ray crystallography, site-directed mutagenesis and functional studies. Locations of the binding sites and results of the functional studies were used to draw conclusion on molecular mechanisms of protection afforded by MntC: antibodies belonging to two of the groups are predicted to interfere with Mn2+ transfer from the protein to the transmembrane channel pore, while the third group of the antibodies is expected to interfere with Mn2+ binding to MntC itself. The net result in both cases is impaired Mn2+ transport across the bacterial membrane and increased susceptibility of the bacterium to the oxidative stress, likely due to the reduced activity of superoxide dismutase which requires Mn2+ as an essential co-factor for activity. Staphylococcus aureus protein MntC is the ligand-binding component of the ABC-type manganese transporter MntABC, which is at least partially responsible for the organism’s resistance to the oxidative stress , Amax is absorbance at saturation and Kd is the dissociation constant.Microtiter plate wells were coated overnight with 2 μg/mL of wild type MntC, MntC-pLH94 or BSA control (100 μL/well). Wells were subsequently blocked with 200 μL /well of 2% Nonfat Dry Milk in PBS, pH 7.4, 0.05% Tween-20 (blocking solution) for 60 min at room temperature. Serial dilutions of mAB 305-78-7 (100 μL each) were added to the wells and incubated at 37°C for 2 hours. Plates were then washed 3 times with the blocking solution (200 μL /well) and incubated for 60 minutes at 37°C with alkaline phosphatase-conjugated goat anti-mouse IgG . Immunocomplexes were detected via absorbance measurements at 405 nm using 1-Step pNPP  chromogenic substrate (100 μL /well). Dissociation constants were determined from fitting of the absorbance data to the equationKD = koff/kon).Binding affinity of mAB 305-78-7 to recombinant mutant MntC proteins was measured with a ForteBio Octet HTX instrument . Experiments were conducted in 96-well black opaque plates  with 30°C incubation temperature and 1000 rpm agitation. To ensure proper immersion of the biosensors, each well contained a final volume of 200 μL. Anti-murine IgG Fv biosensors  were saturated with mAB 305-78-7 and then incubated with two-fold serial dilutions of recombinant MntC proteins. Duplicate measurements were captured within each experiment by reusing the reagent samples with fresh biosensor tips. Kinetic measurements were repeated two times in independent experiments to determine average values. Data acquisition and analysis was performed with Octet software version 8.2 . Buffer-subtracted kinetic data were globally fitted with a 2:1 Langmuir model to obtain a dissociation rate constant  have been published  and willAntibody binding epitopes are identified through comparison of the deuterium accumulation plots for each peptide obtained with and without the antibody present. Conclusions on decreased deuterium accumulation in the presence of the antibodies (or lack thereof) were drawn based on the combination of multiple criteria: (1) significant difference in deuterium accumulation (>10% of the maximum number of deuterons that can be exchanged onto a given peptide) between the protein alone and the protein-antibody complex, (2) qualitative agreement between data from multiple time points for the same peptide, (3) quantitative agreement between data obtained for multiple charge states of the same peptide (when available), and (4) consistency of the data obtained from multiple overlapping peptides covering the same part of the protein sequence. It should be noted that each peptide “reports” on the exchange that is happening starting with amino acid #3 of that peptide, since the exchange on the two N-terminal amino acids is instantaneous and is not informative . For exaIn some cases, it appears that a peptide may be exchanging more protons than physically possible. One has to keep in mind, that the fully deuterated sample used to determine Mw(FD) from Functional antibody mAB 305-78-7 belongs to the interference group 2 , while E247 potentially forms a salt bridge with K58 on the heavy chain . In an a2+ binding to the protein. A decrease or lack of Mn2+ binding as a result of these amino acid substitutions distal from the Mn2+ binding site would indicate that global protein structure has been significantly disrupted by the mutations. The results of the ITC experiments are shown in 2+ binding by MntC-pLH94 are essentially identical to those of the wild type protein, providing additional evidence that the H234F, E247L and K254M substitutions had no effect on the global structure of MntC.Folding state of MntC-pLH94 was confirmed via far- and near-UV CD spectroscopy. CD spectra of MntC-pLH94 are shown in d was 1.2 nM, and the binding enthalpy was -19.9 kcal/mol. No binding was observed in the case of MntC-pLH94 (Binding of mAB 305-78-7 to MntC-pLH94 was tested using ITC and Enzyme-Linked Immunosorbent Assay (ELISA), as described in Materials and Methods. ITC experiments showed that binding of the wild type MntC to mAB 305-78-7 was stoichiometric , indicattC-pLH94 , indicatTo confirm that individual residue-residue interactions seen in the crystal structure indeed play roles in mAB 305-78-7 binding to MntC in solution and to characterize individual contributions of H234, E247L and K254 to the complex formation, we have additionally cloned and expressed a series of MntC variants containing substitutions at those positions. No structural characterization was done for those variants, since no structural changes could be detected in the triple mutant described above, therefore it is reasonable to expect that single mutants would have no effect either. Binding of these mutants to the antibody was characterized using Bio-Layer Interferometry on an Octet HTX instrument, as described in “Materials and Methods”. The results are shown in S. aureus infection, we studied Mn2+ binding to the protein complexed with the antibodies. The results are shown in 2+ binding to MntC is stoichiometric under experimental conditions used, we will not draw conclusions on the association constants derived from these experiments. It should be noted, however, that stoichiometries and binding enthalpies in the experiments with MntC bound to mAB 305-72-5 or mAB 305-101-8 are very comparable to those derived from the control experiment with free MntC, suggesting that these two antibodies do not affect Mn2+ binding by the protein. This is not the case with MntC bound to the Fab fragment of mAB 305-78-7. Although the first Mn2+ injection does produce a noticeable heat exchange  was designed to further confirm mAB 305-78-7 epitope identification. We have chosen two residues that could potentially form intermolecular hydrogen bonds , along with a residue that appears to form an intermolecular salt bridge (E247). Our goal was to minimize the number of amino acid substitutions to minimize any potential disruption or alteration of MntC structure, hence only three out of five residues with side chains involved in intermolecular interactions were selected. Substitutions at these three positions were sufficient to completely disrupt binding, as evidenced by the ITC and ELISA results Figs  and 7. T protein . Taken tS.aureus strains susceptible to the oxidative stress. Considering that manganese, which is the metal transported by MntC, is an essential co-factor of superoxide dismutases, we hypothesized that antibody binding could inhibit MntC-Mn2+ interaction or interfere with Mn2+ transport through the channel pore, making bacteria more susceptible to the attack by neutrophils. A recently published paper by Ahuja and co-workers [2+ transfer from MntC to the importer MntB. FabC1 and our monoclonal antibody mAB-101-8 have overlapping epitopes, thus, they both belong to the interference group 3 as defined by Anderson et al.[2+ release into the channel pore. mAB-305-72-5 binding epitope lies on the same face of MntC molecule as that of mAB-101-8, although on the N-terminal lobe of the structure. Binding of the antibody to this site therefore would also be expected to sterically interfere with MntC-MntB interaction, similar to mAB 305-101-8 or FabC1 interaction, once again preventing Mn2+ transfer into the channel pore.The results reported in this work shed light on the molecular mechanism of protection afforded by the anti-MntC antibodies. Another report from our laboratories  demonstr-workers  providedon et al.. Thus it2+ binding site solvent accessible, while folding of these residues into an alpha-helix in another molecule of the asymmetric unit blocked solvent accessibility. It is tempting to speculate that binding of mAB 305-78-7 to residues 240–258 would stabilize the alpha-helix and, correspondingly, prevent binding of Mn2+ to the protein. This is indeed what seems to be happening. ITC titration of MntC bound to the Fab fragment of 305-78-7  and 3  prevent transport of the Mn2+ ions that were already acquired across the bacterial membrane. The net result of the impaired manganese transport would be reduced activity of superoxide dismutase and increased susceptibility of the pathogen to oxidative stress that the organism will encounter after opsonophagocytosis by the neutrophils. The protective antibodies that belong to these interference groups will effectively starve the bacterium of Mn2+ that it needs for survival.Taken together, experimental data reported here, along with results from Anderson et al., Handke S.aureus infection, however they are rapidly generated as a result upon infection indicating that the antigen is expressed and exposed early in infection [In conclusion, we would like to add that the results reported in this work provide deeper understanding of the MntC antigen mechanism of action within SA4Ag. Investigational clinical studies demonstrated that antibodies that compete with these functional monoclonal antibodies are not detected in humans without documented nfection . IdentifS1 FigX-axis—time, y-axis—number of deuterons exchanged at a given time point. The plots are scaled to the maximum number of deuterons that can be exchanged onto the backbone of the peptide. Blue symbols and lines—deuterium accumulation on MntC alone, red symbols and lines—deuterium accumulation on MntC in the presence of the antibody. When available, multiple plots shown for the same peptide correspond to the different charge states of the peptide.(PDF)Click here for additional data file.S2 FigX-axis—time, y-axis—number of deuterons exchanged at a given time point. The plots are scaled to the maximum number of deuterons that can be exchanged onto the backbone of the peptide. Blue symbols and lines—deuterium accumulation on MntC alone, red symbols and lines—deuterium accumulation on MntC in the presence of the antibody. When available, multiple plots shown for the same peptide correspond to the different charge states of the peptide.(PDF)Click here for additional data file.S3 FigX-axis—time, y-axis—number of deuterons exchanged at a given time point. The plots are scaled to the maximum number of deuterons that can be exchanged onto the backbone of the peptide. Blue symbols and lines—deuterium accumulation on MntC alone, red symbols and lines—deuterium accumulation on MntC in the presence of the antibody. When available, multiple plots shown for the same peptide correspond to the different charge states of the peptide.(PDF)Click here for additional data file.S4 FigFar-UV (panel A) and near-UV (panel B) CD spectra. Black—spectra of the wild type protein, red—spectra of MntC-pLH94.(PDF)Click here for additional data file.S5 Fig2+ binding studies conducted with wild type MntC (Panel A) and MntC-pLH94 (Panel B) are illustrated. Upper panels show experimental heat flow and lower panels show the integrated heat of each individual injection (symbols). Solid lines in the lower panels show fits of the experimental data to the “single class of binding sites” model.Mn(PDF)Click here for additional data file.S6 FigPanel A–experiments with mAB 305-78-7, panel B–experiments with mAB 305-101-8. Open circles: integrated heats of the corresponding peptide injections into the ITC cell containing an appropriate antibody, filled circles—integrated heats of the full length MntC injections into the ITC cell containing either mAB 305-78-7 or mAB 305-101-8. Solid lines—fits to the “single class of binding sites” model. mAB 305-78-7 titration with the synthetic peptide was aborted after 7 injections when it became evident that no heat exchange and, therefore, no interaction is taking place.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Tablea—association constant, ΔH—enthalpy change upon binding, ΔS—entropy change upon binding, Kd—dissociation constant.N—interaction stoichiometry, K(PDF)Click here for additional data file.


Having authored two major ophthalmology textbooks and honored with the eponym, the “Dalrymple sign,” many are familiar with the works of Dr. John Dalrymple when it comes to the eye. However, few are aware of his other, numerous and wide-ranging contributions to the fields of science and medicine. In this article, we discuss the life and work of a man dedicated to the pursuit and advancement of knowledge and education. Many are familiar with the works of Dr. John Dalrymple when it comes to the eye because he was one of the pioneers of medical education within the field of ophthalmology. Having authored two beloved and well-referenced textbooks, "The Anatomy of the Human Eye" and "The Pathology of the Human Eye," Dalrymple is remembered as an expert in his field and is commemorated with the eponym, “Dalrymple’s sign,” used to describe the exophthalmos seen in Grave’s disease . HoweverDalrymple was very active in the scientific and medical communities, frequently lending his expertise to colleagues, participating in several scientific societies, and even, at times, invoking heated debates among his peers. In this article, we discuss Dalrymple’s life and his many contributions.The Anatomy of the Human Eye—a 300-page account of the history and current knowledge of the eye, with five plates of illustrations and accompanying explanations [Dalrymple was born in the city of Norwich in 1803 as the eldest son of Dr. William Dalrymple, a surgeon. John followed in his father’s footsteps, attended Edinburgh University, and became a member of the Royal College of Surgeons in 1827. After settling in London and designating ophthalmology as his field of choice, Dalrymple was elected assistant-surgeon at the Royal London Ophthalmic Hospital in 1832. Two years later, Dalrymple published his first book, anations -14.Within the book preface, Dalrymple explains his reason for writing the book :“During this period, I delivered to the pupils of the London Ophthalmic Infirmary a course of lectures upon the anatomy of the human eye, accompanied by a series of dissections laboriously and minutely followed out. For the purposes of these lectures many authors were of course consulted, and the knowledge contained in their respective works selected and condensed. In my search for authorities, I found no work in the English language, especially dedicated to the anatomy of the organ of vision…”“…very numerous monographs on separate and particular structures of that organ are to be found, either published in distinct form, or scattered through our scientific journals. But written at very different periods, and almost wholly without system or arrangement, it is difficult, at least for students to avail themselves of the valuable treasures they contain. Under these circumstances, I have been induced to combine in a monographic form the many discoveries and improvements that have of late years enriched the department of anatomical sciences.”In the following years, Dalrymple published several scientific articles and became a full surgeon. Scientific publications during this time clearly refer to Dalrymple as an active member within the scientific community. As an avid researcher, Dalrymple frequently published original articles and letters to the editor in response to various new developments -18. As eAs is typical within any lively community, conflicts often arise. In one instance, Dalrymple incited a heated exchange with Dr. WC Wallace of New York because Dalrymple implied that the idea for Wallace’s paper was taken from his demonstration :“In a number of the American Journal of Science and Arts, will be found a somewhat similar account of a muscle, discovered in the eye of the streaked bass…by Mr. W. Clay Wallace, surgeon to the New York institution for the blind. This gentleman did me the favor to send me over, about twelve months since, his paper published in that journal. From the circumstance of my not being aware of being personally acquainted with Mr. Wallace, I cannot help suspecting that he is one of the Americans to whom the observations made by me, were imparted at the ophthalmic hospital, some years ago. I beg, however, to return him my thanks for much new and interesting matter, which has been added by him.”Wallace did not take kindly to this accusation and wrote a rebuttal to Dalrymple’s letter :“I am not an American, therefore the gentleman thus roundly accused are fully exculpated. As there was nothing on the subject in Mr. Dalrymple’s book, a copy of my paper was forwarded to him soon after his publication appeared. If disposed to deviate so far from candour and courtesy as he has done, I might say, with far more plausibility, I cannot help suspecting that is one of those to whom the observations made by me were imparted through the medium of ‘Silliman’s Journal’ in 1834, and now published as his own in the ‘Magazine of Natural History’ in 1838… I have demonstrated the way in which the eye is adjusted to distances in different animals, to many gentlemen of the highest rank in the profession, and there was no difference of opinion about the structure and uses of the various parts of the organ. An attentive examination is all that is required to make the adjustment of the eye as plain as any other truth in physiology.”Upon reading Wallace’s rebuttal, Dalrymple responded by insisting on the originality of his work and apologizing for his accusation :“…Allow me space for a few observations, in order that I may put myself right with regard to the somewhat angry remarks of Mr. Clay Wallace, of New York… That I should, however, have conceived that Mr. Wallace, residing in, and attached to one of the public establishments of, New York, was an American either by birth or adoption, was at least a very pardonable mistake, and one that involved him no disgrace. He has, however, retaliated on me by suspecting that I had read and copied, without acknowledgement, the observations made by him in Silliman’s Journal in 1834. My answer is that I have never seen that Journal, nor did I know anything of Mr. Wallace’s dissections… And further, that my dissections were made two years or more previous to the date of Mr. Wallace’s paper in Prof. Silliman’s Journal.…as I find Mr. Wallace has distinctly denied, in a recent number of Silliman’s Journal, having either seen or heard of my observations, I am bound in justice to relieve him from any imputation, which I am now sorry I have made. While anxious to clear myself from the suspicion of plagiary, I can do no less than withdraw that portion of my charge which relates to Mr. Wallace…”Lancet 1841, he discusses the contents of Ferrall’s paper, “On the Anatomy and Physiology of certain Structures in the Orbit not previously described,” and how its claim of a new discovery, “not hitherto been noticed by anatomists,” had already been covered in his book, The Anatomy of the Human Eye. Dalrymple went on further to explain [Rightly so, Dalrymple was proud of his achievements and did not shy away from claiming credit when it is due. In another letter to the editor, published in the  explain :“The only difference between this description in 1834, and the novelties claimed by M. Bonnet and Mr. Ferrall, is, that I have described the capsule as cellular and they as fibrous; while, upon a careful review of the question, I still maintain that this tunica vaginalis (a term I have no objection to) is composed of condensed cellular membrane, and by no means a true fibrous tissue in the ordinary sense of the word. It is a little too much fashion of the present day to forget the labours of those who have gone before, and that custom lends force to the old adage, ‘Omnis noveltas nil nisi oblivion.’”As evidence of Dalrymple’s reputation, the editors responded to his letter by praising Dalrymple’s book and being inclined toward Dalrymple’s claims .Dalrymple was undoubtedly even more involved in the fields of medicine than his publications reflect, as papers during this time seem less stringent about correctly acknowledging and citing collaborators. Several papers mention Dalrymple’s name within the text, yet provide little detail on his exact contributions -26.Perhaps one of the most unique contributions that Dalrymple offered was his drawings. There are numerous examples of his works in various books and journal articles. These intricate pieces reflect his skill as a microscopist and his patience and dedication toward the advancement of science.In 1846, Dalrymple’s collaboration with Dr. Henry Bence Jones elucidated the skeletal defects found in patients with multiple myeloma. In “On the microscopial character of mollities ossium,” Dalrymple conducted and documented the post-mortem examination of Mr. Thomas Alexander McBean, the first well-documented case to be diagnosed with multiple myeloma ,10. Exam“The disease appears to have commenced in the cancellated structure of the bone, for the external osseous laminae are firmer and more healthy than the internal. The smooth surface of the rib, however, is raised by internal growths elevating the outer laminae here and there, into irregularly-sized and rounded dark red projections, visible through the periosteal covering. The outer layers are still hard, requiring the exertion of some force to cut them; they are thin, however, and when sliced, expose large cancellous cavities filled by a red gelatiniform substance, threaded here and there by fine bony fibres.” Having accomplished so much in his career, it is hard to believe Dalrymple was chronically plagued by ill health. Dalrymple was sick so often that he frequently could not attend his surgeries, and in 1847, an additional assistant surgeon was hired specifically to assist with his workload .The Pathology of the Human Eye, was published just before his death. The book contained 36 watercolor paintings of the eye by various artists [Edinburgh Medical and Surgical Journal [Having had poor health his entire life, Dalrymple passed away on May 2, 1852, at the age of 49, due to renal disease . His sec artists . Before  Journal :“In these engravings, Mr. Dalrymple undertakes to illustrate by coloured drawings the principal disease of the eye and its appendages. He does not profess, he states, to give a systematic treatise on the subject, but to illustrate in a series of drawings, the various forms of ophthalmic disease as they occur in nature with such explanations as may adequate to identify them with the symptoms, and with the general treatment of the case……Upon the whole, these drawings possess qualifications which recommend them strongly to the attention of the surgeon.Provincial Medical and Surgical Journal [In the  Journal :“…Mr. Dalrymple has, we think, judged rightly in throwing the results of his experience into the present form. His work thus becomes illustrative of any one or all of these systematic treatises, and indeed makes frequent reference to many of them for details of symptoms and treatment; while, through its admirable couloured drawings, it at once brings the objected symptoms prominently forward, and characterizes and defines them in the accompanying text in such a manner as to render the work most valuable for study and consultation……Of the manner in which the drawings are executed and coloured, it is scarecely possible to speak too highly; they are beautiful and characteristic represenations of the diseases which they illustrate, and the entire work, if completed in the same style of excellence, cannot fail to give the highest satisfaction to those who may become possessed of it.”British and Foreign Medical Chirurgical Journal [In the  Journal :The Pathology of the Human Eye’ now before us, is perhaps the most interesting that has yet appeared, as regards alike the subjects treated, and the mode in which they are handled, both by the author, and the artists who have executed his illustrations …”“The fasciculus of Mr. Dalrymple’s ‘The Pathology of the Human Eye, as an exhaustive search of all of Dalrymple’s published works resulted in no separate accounts or case reports on this topic. In 1849, Cooper published “On protrusion of eyes,” mentioning Dalrymple’s extensive experience and pathophysiologic explanation for this ophthalmologic sign. In this paper, there is no use of the eponym either, lending evidence to the fact that it manifested later, possibly after Dalrymple’s death. Cooper offers Dalrymple’s explanation in the following excerpt:The eponym “Dalrymple’s sign” is likely to have originated after the publication of the book, “The prominence of the eyes is probably due to the operation of two causes. An absence of the proper tonicity of the muscles by which the eyes are retained in their natural positions in the orbit; and some amount of venous congestion of the tissues forming the cushion behind the globes.” In his final will and testament, Dalrymple, staying true to his role as an educator, bequeathed his drawings of diseases of the eye for the use of the students at the Royal Ophthalmic Hospital. Per James’ biography of Dalrymple, over 70 years after Dalrymple’s death, “Those beautiful paintings of diseases of the eyes are still preserved in the library of the Royal London Ophthalmic Hospital” .In his relatively short career, John Dalrymple contributed greatly to the fields of science and medicine. Having authored two of the earliest and most comprehensive texts on ophthalmology, as well as numerous journal articles, Dalrymple is well-deserving of an eponym to honor his work. Had he lived a longer, healthier life, Dalrymple would have undoubtedly produced further works of art and scientific advancements.


Aspergillus nidulans grown on cereal starches from wheat and high-amylose (HA) maize, as well as legume starch from pea for 5 days.Starch is the second most abundant plant-derived biomass and a major feedstock in non-food industrial applications and first generation biofuel production. In contrast to lignocellulose, detailed insight into fungal degradation of starch is currently lacking. This study explores the secretomes of Aspergillus nidulans grew efficiently on cereal starches, whereas growth on pea starch was poor. The secretomes at days 3–5 were starch-type dependent as also reflected by amylolytic activity measurements. Nearly half of the 312 proteins in the secretomes were carbohydrate-active enzymes (CAZymes), mostly glycoside hydrolases (GHs) and oxidative auxiliary activities (AAs). The abundance of the GH13 α-amylase (AmyB) decreased with time, as opposed to other starch-degrading enzymes, e.g., the GH13 AmyF, GH15 glucoamylases (GlaA and GlaB), and the GH31 α-glucosidase (AgdE). Two AA13 LPMOs displayed similar secretion patterns as amylolytic hydrolases and were among the most abundant CAZymes. The starch-active AnLPMO13A that possesses a CBM20 carbohydrate-binding module dominated the starch-binding secretome fraction. A striking observation is the co-secretion of several redox-active enzymes with the starch-active AA13 LPMOs and GHs, some at high abundance. Notably nine AA9 LPMOs, six AA3 sub-family 2 (AA_2) oxidoreductases, and ten AA7 glyco-oligosaccharide oxidases were identified in the secretomes in addition to other non-CAZyme oxidoreductases.The co-secretion and high abundance of AA13 LPMOs are indicative of a key role in starch granule deconstruction. The increase in AA13 LPMO abundance with culture time may reflect accumulation of a more resistant starch fraction towards the later stages of the culture. The identification of AmyR sites upstream AA13 LPMOs unveils co-regulation of LPMOs featuring in starch utilization. Differential deployment of amylolytic hydrolases and LPMOs over time suggests additional regulatory mechanisms. The abundant co-secretion of distinct AA3 and AA7 oxidoreductases merits further studies into their roles and possible interplay with LPMOs and other enzymes in the deconstruction of starchy substrates. The study reports for the first time the biological significance of LPMOs in starch degradation and the temporal interplay between these and amylolytic hydrolases.The online version of this article (doi:10.1186/s13068-016-0604-0) contains supplementary material, which is available to authorized users. Starch is one of the most abundant renewable biopolymers in nature . AnnuallThe high utilization potential of starch as a renewable biological resource and an industrial feedstock motivates efforts to improve starch hydrolysis yields, particularly from more resistant starch types and for shortening process times. Gain in yields of hydrolysis would have a considerable impact on efficiency and cost of industrial starch processing as well as the reduction of environmental impact of this process .The classical paradigm of polysaccharide degradation by hydrolytic enzymes, which has been valid for decades, was recently re-visited by the discovery of the copper-dependent lytic polysaccharide monooxygenases (LPMOs) , 7. LPMOAspergillus nidulans that is taxonomically related to well-established industrial cell factory species such as Aspergillus niger and Aspergillus oryzae [A. nidulans to sustain growth on three different starches in the course of 5 days. The data demonstrate differences in growth and secretomes on the selected starch substrates. A common feature of growth on starch was that two AA13 LPMOs including the modular starch-specific enzyme joint to a starch-binding domain of family 20 (CBM20) were among the most abundant CAZymes together with a variety of LPMOs and other oxidative enzymes. This finding suggests that oxidative cleavage of α-glucosidic bonds plays a significant role in starch breakdown. Altogether, the novel insight into enzymatic activities secreted by A. nidulans and related fungi for efficient starch breakdown is relevant for design of enzyme mixtures with enhanced bioconversion efficiencies of starches especially those resistant to hydrolytic degradation.Filamentous fungi secrete impressive amounts of hydrolytic enzymes targeting polysaccharides. In addition to glycoside hydrolyses (GHs), several fungi also produce a multitude of LPMOs , 21. In s oryzae . By inteA. nidulans to sense differences in the origin and structure of the starch substrates and to fine-tune the composition of secreted enzymes accordingly, this fungus was grown on wheat, high-amylose (HA) maize, and pea starches, and the resulting secretomes were analyzed. Aspergillus nidulans grew efficiently on wheat and HA maize starch and no intact starch granules were distinguished from the fungal biomass after 5 days, suggesting extensive degradation of both starches. By contrast, growth was poor on pea starch, leaving significant amounts of intact starch granules at culture harvest, which clearly demonstrates important differences due to the botanical origin and properties of the starch on enzymatic deconstruction and growth.To assess the ability of The α-amylase and α-glucosidase activities were measured in the filtered culture supernatants. The average activities of the biological triplicates in different starch media at days 1–5 are shown in Fig. The α-glucosidase activity increased over time in all samples and showed the highest activity (53 U/ml) in HA maize starch medium on day 5 Fig. b. The glA. nidulans cultures grown on wheat, HA maize, and pea starch media were analyzed using liquid chromatography combined with tandem mass spectrometry (LC-MS/MS). The analysis of the data set  represented the largest category  from certain CAZy families, e.g., GH3, GH16 and GH43, and CE10 as well as AA3, AA7, and AA9, with the three latter families harboring oxidative activities  were the most abundant CAZymes in all samples  seemingly at higher abundance than AnLPMO13A (Q5B1W7). The N-terminal histidine residue of both these AA13 enzymes seemed to be methylated in virtually all samples  based on the identification of the “H(Me)GYLTVPASR” peptide (best MASCOT score = 78) that is identical in both enzymes, while the non-methylated counterpart could not be detected. Notably, the abundance of both proteins increased slightly over time , three of which  were highly abundant in most samples, the two former and the latter showing a decreasing and increasing trend over time, respectively  is Catalase B , which also shows a slight decrease in abundance over time in wheat and maize, and an opposite trend in pea . α-Arabinofuranosidases of GH54 (O74288) and GH62 (Q5AUX2) were also identified, the latter only in wheat and HA maize starch media together with a GH53 β-1,4-endogalactanase (Q5B153). Moreover, a putative GH93 exo-arabinanase (Q5BBM0), a GH27 (Q5AX28) and a GH36 (Q5AU92), both the latter being α-1,6-galactosidases were identified. Putative pectin-degrading enzymes including a GH2 β-glucuronidase (Q5BAN5), a GH105 unsaturated rhamnogalacturonan hydrolase (Q5AQP7), and two putative GH28 exo-polygalacturonases (Q5ASG9 and Q873X6) were detected, the latter only in HA maize starch cultures. Finally, a GH35 putative β-galactosidase (Q5BFC4) was detected in HA maize and pea starch cultures.Ten putative β-glucoside and cellulose-degrading enzymes were identified. A GH1 (Q5AR97) and five putative GH3 β-glucosidases  were identified in all samples. Two of these enzymes  appeared only after 4 days in the pea starch. Notably, cellobiohydrolases (GH6) and endoglucanases (GH5_7) active on cellulose were not identified in the pea starch culture supernatant. Two GH5  were only present in the maize, whereas the GH6 (C8VSG6) and GH7 (Q5B7R2) were also identified in wheat starch cultures. Twelve putative hemicellulose-degrading enzymes were identified including a GH10 (Q00177) and a GH11 (P55332) β-1,4-endoxylanases and four putative GH43 β-xylosidases/α-N-acetylglucosaminidase (G5EB27). The putative GH71 α-1,3-glucanase (C8VKQ6) was detected only on days 4 and 5 in all cultures. In addition, three α-mannan-degrading enzymes, one GH47 (Q5BF93), and two GH92 (C8VSU5 and Q5BAV5) were identified.Fifteen secreted enzymes putatively involved in fungal cell wall modification and degradation were identified including six GH16 glycanases , a GH17 β-1,3-endoglucanase (Q5AUT0), two putative GH55 β-1,3-exoglucanase (Q5B3Q5 and C8VQV2), three GH72 β-1,3-glucanosyltransferases , a GH81 endo-β-1,3-endoglucanase (C8VT57), and a putative GH20 A. nidulans secreted enzymes that possess affinity for starch. The elution fraction from this purification was analyzed by SDS-PAGE . The bulk of extracted starch from these crops is used for non-food industrial applications including first generation biofuel that still contributes more than two thirds of the total biofuel production [The abundance of starch from terrestrial biomass is only surpassed by cellulose. An excess of two billion tons of starch are annually produced from cereals and coarse grains and another 700 million tons from roots and tubers  and GH15 glucoamylases  is among the top four most abundant proteins at all time points in the wheat and maize starch cultures , which has a CBM20 appended, is also abundant in the secretomes of cereal starches . The abundance of AnLPMO13A may be underestimated as compared to AnLPMO13B, due to the preferential CBM20-mediated binding of the former onto granular starch , indicating that they also share substrate specificity. A notable difference is that AnLPMO13B possesses a 75 amino acid C-terminal extension of unknown function as compared to the long linker and the C-terminal CBM20 of AnLPMO13A. The absence of AnLPMO13B in secretome fraction captured by β-cyclodextrin affinity suggests that the affinity of this enzyme to insoluble starch is much lower than AnLPMO13A that possesses a CBM20. These differences indicate that the AA13 LPMOs may target different parts of the starch structure, yielding complementary activities. This has been observed for the conserved cellulose-targeting LPMO pair encoded by Streptomyces coelicolor, which contributed synergistically when combined in cellulose degradation [Several studies investigating the secretomes of plant biomass-degrading microorganisms have reported the abundant presence of LPMOs, indicating their importance in biomass conversion , 29. HowLPMO13B QB027 is aLPMO13B QB027 is ad starch , whereasradation . Interesradation . This hiA. nidulans genome were not represented in the secretomes  [AoAA11), it is possible that the function is related to fungal cell wall remodeling.One of the two AA11 LPMOs is observed in the secretomes, albeit at low abundance. The enzyme shows only moderate similarity to the chitin-degrading 1 (51 %) , precludIt is well established that the sub-family 1 members of the glucose, methanol, choline (GMC) oxidoreductases (AA3s), i.e., the cellobiose dehydrogenases (CDHs), can provide electrons to LPMOs , 46. Mal2O2. A possible role for enzymes generating H2O2 is to provide lignin-degrading enzymes like laccases and peroxidases with hydrogen peroxide, required for lignin depolymerization. On the other hand, no or little lignin is present in the substrates provided in the present experiments, possibly indicating a different role of these enzymes. Biochemical characterization of the identified AA7 and AA3 enzymes is warranted to verify their roles.Both aryl-alcohol oxidases and glucose dehydrogenases, both assigned into AA3_2, have been, very recently, shown to be able to transfer electrons to LPMOs , 48. Two2O2 generated from the activity of AA3 and AA7 oxidases on products derived from depolymerization of starch (or other components in the substrate matrices) provides a rationale for the dominance of catalase B (CatB) in most secretomes. Catalase catalyzes the conversion of H2O2 to O2 and H2O, thus protecting the fungus from H2O2 toxic effects . Indeed, the expression of CatB has previously been demonstrated to be induced by H2O2 or H2O2-generating conditions [2O2 production [The Hnditions , and theoduction . A seconoduction . Thus, ioduction .amyA, amyB, and amyF, and the glucoamylase glaB genes in A. nidulans [A. nidulans genome revealed the presence of the AmyR consensus sequence 5′-CGGN8CGG-3′ at around −300 basepairs (−318 to −305) in the promotor region of the AnLPMO13A and around −600 basepairs (−612 to −609) for AnLPMO13B. This is the first time a common regulatory link has been identified between GHs and LPMOs. Interestingly, hierarchical clustering analysis revealed that both AA13 enzymes have very similar secretion patterns as distinct amylolytic enzymes under the control of AmyR  before resuspension in autoclaved growth media or as substrates for enzymatic assays. p-Nitrophenyl-α-d-glucopyranoside (PNPG) and p-nitrophenol (PNP) were from Sigma-Aldrich, and insoluble Blue Starch was a custom preparation from Pharmacia .The wheat starch was from Sigma-Aldrich , pea and high-amylose (HA) maize starches were from KMC . The starches for growth experiments were not autoclaved to minimize changes of the native starch granule structure, but washed twice in 70 % ethanol and water and subsequently pelleted by centrifugation , was pre-grown on malt extract agar plates containing 1 % wheat, HA maize or pea starches for 5 days at 30 °C until new mycelia were formed. Mycelial plugs were used to inoculate 1 L of minimal medium containing 1 % (w/v) carbon source with a start pH of 6.5 in 3 L baffled shake flasks. The minimal medium contained, per liter, 6 g NaNO3, 0.52 g KCl, 0.52 g MgSO4·7H2O, 1.52 g KH2PO4, and 2 ml of Hutner’s trace elements. Hutner’s trace elements contained, per liter, 2.2 g ZnSO4·7H2O, 1.1 g H3BO3, 0.5 g MnCl2·4H2O, 0.5 g FeSO4·7H2O, 0.16 g CoCl2·6H2O, 0.16 g CuSO4·5H2O, 0.11 g (NH4)Mo7O24·4H2O, and 5 g EDTA. The cultures were incubated on a rotary shaker (150 rpm) at 30 °C for 5 days. All experiments were performed in biological triplicates. The medium was supplemented with antibiotics (100 µg/ml ampicillin and 34 µg/ml chloramphenicol) to inhibit growth of possible bacterial contamination from the starch substrates.Both enzymatic activities were measured and averaged from the biological triplicates.g, 3 min) 200 µl supernatant was transferred to a 96 well microtiter plate and A620 values were used to determine enzyme activity. One activity unit (U) was defined as the amount of enzyme that leads to an increase in A620 of 1 absorbance unit in the reaction mixture under these experimental conditions.α-amylase activity of the fungal cultures was assayed toward insoluble Blue Starch (iBS). The activity was measured using iBS (6.25 mg/ml) suspended in 10 mM MES, pH 6.5. The reaction mixture (900 µl) was incubated for 15 min at 37 °C after addition of the culture filtrate (100 µl). The reaction was stopped by addition of 0.5 M NaOH (200 µl). After centrifugation (4000×p-nitrophenyl α-d-glucopyranoside (PNPG) as the substrate. The activity in culture filtrates was assayed toward 2 mM PNPG in 10 mM MES, 0.005 % Triton X-100, pH 6.5 and 10 μl culture supernatant in 50 μl reactions, at 37 °C for 60 min. The reaction was stopped by adding 1 M Na2CO3 (200 μl). The amount of released p-nitrophenol (PNP) was measured spectrophotometrically at A410 using PNP (0–2 mM) as a standard. One unit of activity (U) was defined as the amount of enzyme that released 1 μmol/min of PNP at the given conditions.The α-glucosidase activity was determined using g, 15 min, 4 °C) and filtration  and stored at −20 °C until further use. Samples (10 ml) from three biological replicate cultures were collected from day 3 to 5 under sterile conditions. Protein concentrations of the culture filtrates were determined using a Bradford assay  with a BSA standard according to the manufacturer’s instructions.Culture filtrates containing the fungal secretomes were separated from mycelia and residual insoluble starch by centrifugation  to pellet precipitated protein and washed with 300 µl ice-cold 10 mM HCl in 90 % acetone. After centrifugation , pellets were dried, redissolved in 100 µl 20 mM Tris–HCl at pH 8, reduced with 10 mM  dithiothreitol (DTT) and incubated at room temperature for 30 min. Alkylation was performed by adding 15 mM  iodoacetamide (IAA) and incubating at room temperature for 30 min in the dark. The proteins were digested with 20 µl 12.5 ng/µl sequencing-grade modified trypsin  and incubated at 37 °C for 16 h. Trypsination was stopped with 0.5 %  trifluoroacetic acid (TFA). Samples were dried in SpeedVac and purified with ZipTip C18 pipette tips  according to manufacturer’s instructions. Purified samples were dried and dissolved in 2 % acetonitrile (ACN) and 0.1 % TFA mix.Proteins of culture filtrates (2 ml) were precipitated by direct addition of 500 µl cold 50 % trichloroacetic acid (TCA), incubated at 4 °C for 8 h, centrifuged  and backflushed onto a 50 cm analytical column . At the start, the columns were in 96 % solution A (0.1 % (v/v) formic acid), 4 % solution B (80 % (v/v) ACN, 0.1 % (v/v) formic acid). Peptides were eluted using a 90 min gradient from 4 to 13 % (v/v) solution B in 2 min, 13–45 % (v/v) in 70 min and finally to 55 % B in 5 min before the wash phase at 90 % B, all at a flow rate of 300 nl/min. To isolate and fragment the ten most intense peptide precursor ions at any given time throughout the chromatographic elution, the Q-Exactive mass spectrometer was operated in data-dependent mode to switch automatically between orbitrap-MS and higher-energy collisional dissociation (HCD) orbitrap-MS/MS acquisition. The selected precursor ions were then excluded for repeated fragmentation for 20 s. The resolution was set to R = 70,000 and R = 35,000 for MS and MS/MS, respectively. For optimal acquisition of MS/MS spectra, automatic gain control (AGC) target values were set to 1,000,000 charges and a maximum injection time of 128 ms.The peptides were analyzed in two technical replicates using a nanoHPLC-MS/MS system consisting of a Dionex Ultimate 3000 RSLCnano  connected to a Q-Exactive hybrid quadrupole-orbitrap mass spectrometer (Thermo Scientific) equipped with a nano-electrospray ion source. Samples were loaded onto a trap column  [MS raw files were imported into MaxQuant , 56 versquences)  supplemequences)  includinhttp://csbl.bmb.uga.edu/dbCAN/) [Post-processing was done using Perseus version 1.5.0.31. Proteins categorized as only identified by site and matches to reversed sequences or contaminants were removed. Furthermore, the proteins were filtered so that a valid quantification existed for at least two of the three replicates on at least one substrate. Intensities were log-transformed and missing values imputed based on a downshifted normal distribution. Hierarchical clustering and heat map generation were done with Euclidean distance measure and average linkage. For visualization of trending proteins, ANOVA  was used to filter out proteins that showed no significant change over time. Protein quantitative values were z-scored within substrate and imputed values removed prior to clustering and heat map generation. Secretion prediction was a combination of SignalP , Phobius/dbCAN/) .4)2SO4 to 0.5 M final concentration and agitated until the salt was completely dissolved. Thereafter, the sample was filtrated  and applied to an XK 16/20 column packed with a 20 ml β-cyclodextrin (β-CD) sepharose affinity resin and pre-equilibrated with 4 column volumes of 10 mM sodium acetate buffer, pH 5.5, with 500 mM NaCl at 1 ml/min [The supernatant of the culture grown on wheat starch for 5 days was supplemented with  in the XCell SureLock Mini-Cell system (invitrogen), according to manufacturer’s instructions and visualized by staining with InstantBlue solution . Spots were excised from the visually most prominent protein bands from the stained gel, washed in 300 µl 40 % ethanol at 50 °C for 15 min and in 100 µl 100 % ACN at room temperature for 10 min. The proteins in the gel were reduced with 50 µl 10 mM DTT in 100 mM NH4HCO3 at 56 °C for 45 min. Cysteine alkylation was performed by adding 100 µl 55 mM IAA in 100 mM NH4HCO3 and incubating at room temperature for 30 min in the dark. The gels were washed with 100 µl 50 % ACN, then 100 µl 100 % ACN and dried. The proteins in the gel were digested with 10 µl 12.5 ng/µl sequencing-grade modified trypsin (Promega) dissolved in 10 mM NH4HCO3 and incubated at 4 °C for 45 min, followed by addition of 10 µl 10 mM NH4HCO3 and incubation at 37 °C for 16 h. Samples (1 or 2 µl) were spotted directly onto a MTP AnchorChip target plate , allowed to dry, and overlaid with 1 µl 0.5 µg/µl α-cyano-4-hydroxycinnamic acid (CHCA) matrix in 90 % ACN, 0.1 % TFA. The MS analyses were performed using an Ultraflex II MALDI-TOF/TOF MS . The obtained mass spectra were processed with FlexAnalysis and BioTools software both provided by the instrument manufacturer. Combination of MS and MS/MS data was used as input for databases searching for the spectra from MALDI-TOF/TOF using an in-house-licensed Mascot search engine . Proteins were identified using NCBInr database. The following parameters were set for searching: allowed global modification, carbamidomethyl cysteine; variable modification, oxidation of methionine; missed cleavages, 1; peptide tolerance, 80 ppm; MS/MS tolerance, ± 0.5 Da. The protein identification was considered valid if it matched more than two peptides and the significance threshold for protein identifications was p < 0.05.The eluted sample was analyzed by SDS-PAGE using Novex NuPAGE8CGG-3′ was identified by searching 1000 bp upstream of the start codon of the A. nidulans genes encoding AnLPMO13A and AnLPMO13B. The positions of the 5′ and 3′ ends of the AmyR binding site with putative translational start site as +1 were calculated.The AmyR consensus binding site sequence 5′-CGGN