Nicotiana species in the graft union zone, within a tissue culture system. This has led to the formation of alloploid cells that, under laboratory conditions, gave rise to a novel, alloploid Nicotiana species, indicating that natural grafts may play a role in plant speciation, under certain circumstances.Grafting, an old plant propagation practice, is still widely used with fruit trees and in recent decades also with vegetables. Taxonomic proximity is a general prerequisite for successful graft-take and long-term survival of the grafted, composite plant. However, the mechanisms underlying interspecific graft incompatibility are as yet insufficiently understood. Hormonal signals, auxin in particular, are believed to play an important role in the wound healing and vascular regeneration within the graft union zone. Incomplete and convoluted vascular connections impede the vital upward and downward whole plant transfer routes. Long-distance protein, mRNA and small RNA graft-transmissible signals currently emerge as novel mechanisms which regulate nutritional and developmental root/top relations and may play a pivotal role in grafting physiology. Grafting also has significant pathogenic projections. On one hand, stock to scion mechanical contact enables the spread of diseases, even without a complete graft union. But, on the other hand, grafting onto resistant rootstocks serves as a principal tool in the management of fruit tree plagues and vegetable soil-borne diseases. The ‘graft hybrid’ historic controversy has not yet been resolved. Recent evidence suggests that epigenetic modification of DNA-methylation patterns may account for certain graft-transformation phenomena. Root grafting is a wide spread natural phenomenon; both intraspecific and interspecific root grafts have been recorded. Root grafts have an evolutionary role in the survival of storm-hit forest stands as well as in the spread of devastating diseases. A more fundamental evolutionary role is hinted by recent findings that demonstrate plastid and nuclear genome transfer between distinct  Cucurbitae and Solanaceae species. Thus, the majority of recent grafting research concerns physiological and pathological aspects of vegetable grafting. Grafting also plays an important role in various types of physiological investigations . A broad range of classical grafting techniques can be found in igations , in partigations . An overigations . The hisigations . Undoubtresearch  has openCapsicum annuum L.), old-stage homografts (58 days old plants) had slower graft-take than young-stage (34 days) homografts. Old-stage grafts had a lower percentage of xylem connections and seemed to suffer from drought stress  are presumably always compatible. In heterografts, broadly speaking, intraspecific grafts  are nearly always compatible, interspecific grafts (= rootstock and scion belonging to different species of the same genus) are usually compatible, intrafamilial grafts are rarely compatible, and interfamilial grafts are essentially always incompatible . Examinat stress . The timrequired  but Yin ts e.g., .Graft incompatibility may be defined as failure to form a successful graft union. Yet, despite innumerous follow-ups of graftage in various types of plants, the reasons for graft incompatibility are still vague. Initial healing of the graft union does not in itself ensure long-term compatibility. In cucurbits, apparently successful grafts proved incompatible 25 days after grafting . In certSolanaceae, reciprocal grafts of tomato (Solanum lycopersicum L.) and pepper were considered severely incompatible, whereas tomato and eggplant (Solanum melongena L.) only moderately so, in comparison with compatible tomato homografts (Cucumis melo L.) onto 22 Cucurbitae rootstocks (Cucurbitae species (Castanea) species  onto dwarfing rootstocks  scions and quince (Cydonia oblonga Mill.) rootstocks. Prunasin, a cyanogenic glucoside, rises from the quince rootstock into the pear scion, where it is catabolized enzymatically by pear glycosidase to liberate cyanide at the graft interface. Cyanide ‘poisons’ the graft union tissues, causing cellular necrosis at the graft interface, leading to severe incompatibility (Prunus persica L. Batsch) grafted on myrobalan plum (Prunus cerasifera L. Ehrh) suggested a more general poisoning mechanism, due to progressive impairment of the phloem carbohydrate transport and accumulation of starch above the graft union , which presumably involves some kind of mobile RNA, was one of the pioneering findings in this area , in a distant organ , does not in itself prove its vascular transport, since this compound could also be synthesized in the presumably recipient organ. Therefore, grafting of genetically distinct mutants/transgenes is still being used as a principal means to distinguish between the site of biogenesis and the recipient organs . Roots oagnitude . FurtherSolanum tuberosum L.) tuberization by the graft transmissible miR156 is another recently studied case (Gibberellic Acid Insensitive (GAI) mRNA, including the anticipated changes in plant phenotype  through a variety of tracks, including natural, underground root grafts. This avenue of disease transmission has been underestimated , just asSantalales) and dodder (Cuscuta sp.) transmit pathogens very efficiently, even among intergeneric and interfamilial plant species (Parasitic plants such as mistletoes (= parasitic plants in the order  species . Dodder  species .Persea americana Mill.) transferred a citrus viroid to avocado; the grafted citrus budwood remained viable for almost a year without forming a true graft union (Catharanthus sp.) seems to be unique in its ability to form distant interspecific grafts. The apple proliferation phytoplasma disease was transmitted from infected apple scions to periwinkle rootstocks  rootstocks to overcome the Phylloxera grapevine plague (Citrus tristeza virus (Closterovirus) and other virus-like diseases exploited every possible graft combination  had PGIP activity. PGIP was detected in xylem exudates of untransformed grapevine scions grafted onto transgenic rootstocks expressing pPGIP, indicating that the PGIP protein is graft–transmissible. Such scions revealed improved tolerance toward grapevine diseases caused by Xylella fastidiosa and Botrytis cinerea  also seem to play a role in rootstock-induced disease resistance. Polygalacturonase-inhibiting proteins (PGIPs) are plant cell proteins that specifically inhibit the cell wall degrading endo-polygalacturonases of plant pathogens . Leaf ex cinerea . Followi cinerea . This idFigure 2).Of all grafting issues, the least understood and most controversial is the ‘graft hybrid’ concept. According to this concept grafting may involve stock to scion transfer of genetic material (= graft transformation), leading to heritable changes in the scion. The scion which has acquired certain heritable traits from the rootstock is regarded as a ‘graft hybrid.’ However, graft transformations occur only under ‘Mentor grafting’ conditions, which presumably enforce the transfer of genetic material from stock to scion  are removed throughout the experiment. Stock fruits are also removed in attempt to maximize flow of substances from the stock to the scion . InteresThe graft hybrid concept, which was developed and demonstrated by the Soviet horticulturist Solanaceae, in particular pepper (Within the ‘graft hybrid’ supportive evidence some specific characteristics can be defined. (a) The frequency of the appearance of variant plants is highly variable, sometimes below 1% . Thus, tr pepper , which ir pepper  has beenr pepper  but no fIn their review, Epigenetics refers to reversible heritable changes in genome function that occur without a change in the DNA sequence and may have morphological, physiological, and ecological consequences . ChangesSolanaceae . Root grafting could be induced artificially between potted l months . Naturall months . Althougl months , it may Assessment of the evolutionary significance of grafting involves discussion at two levels: its benefits for plant survival and its potential role in the formation of new species. The plausible ecological benefits of root grafting have been discussed by Addressing the significance of grafting as a potential evolutionary mechanism must take into account the ancient belief that grafting may give rise to new plant species . The ‘grNicotiana species (Recent research has brought us much closer to a potentially real evolutionary grafting mechanism. In their early report  species . Interes“… do not lend support to the tenet of Lysenkoism that ‘graft hybridization’ would be analogous to sexual hybridization. Instead, our finding that gene transfer is restricted to the contact zone between scion and stock indicates that the changes can become heritable only via lateral shoot formation from the graft site.”Nicotiana species was thus obtained, tentatively named Nicotiana tabauca. Still, natural evolutionary formation of new species via this pathway would depend upon natural graft formation and emergence of adventitious lateral shoots, as previously discussed.However, in a more recent report  Bock andNicotiana under tissue culture conditions require further confirmation with other plants in natural settings (The hypothetical possibility of cell fusion graft hybrids has already been mentioned by settings . It is dArabidopsis model has opened the way for meticulous, in-depth grafting research. The currently available molecular tools are expected to advance our understanding and eventually resolve the long standing grafting mysteries.While the technology of grafting has advanced tremendously, the long-term survival of grafted, composite plants is still somewhat unpredictable. The complexity of stock/scion interactions attains a new dimension when pathogenic agents enter the scenery. More enigmatic are the graft hybrid conundrum and the broader evolutionary significance of grafting. The extensive horticultural grafting research has not usually addressed these basic questions. The recently adopted The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Aedes aegypti (L.) is the principal urban vector of dengue viruses. Currently 2.5 billion people are at risk for infection and reduction of Ae. aegypti populations is the most effective means to reduce the risk of transmission. Pyrethroids are used extensively for adult mosquito control, especially during dengue outbreaks. Pyrethroids promote activation and prolong the activation of the voltage gated sodium channel protein (VGSC) by interacting with two distinct pyrethroid receptor sites [Ae. aegypti, a Ile1,016 mutation in the S6 of domain II was discovered in 2006 and found to be associated with pyrethroid resistance in field populations in Mexico. In 2010 a second mutation, Cys1,534 in the S6 of domain III was discovered and also found to be associated with pyrethroid resistance and correlated with the frequency of Ile1,016.Worldwide the mosquito or sites , formed Ae. aegypti collected in Mexico from 2000–2012 to test for statistical associations between S6 in domains II and III in natural populations. We estimated the frequency of the four dilocus haplotypes in 1,016 and 1,534: Val1,016/Phe1,534 (susceptible), Val1,016/Cys1,534, Ile1,016/Phe1,534, and Ile1,016/Cys1,534 (resistant). The susceptible Val1,016/Phe1,534 haplotype went from near fixation to extinction and the resistant Ile1,016/Cys1,534 haplotype increased in all collections from a frequency close to zero to frequencies ranging from 0.5–0.9. The Val1,016/Cys1,534 haplotype increased in all collections until 2008 after which it began to decline as Ile1,016/Cys1,534 increased. However, the Ile1,016/Phe1,534 haplotype was rarely detected; it reached a frequency of only 0.09 in one collection and subsequently declined.A linkage disequilibrium analysis was performed on Ile1,016 and Cys1,534 in vgsc gene requires the sequential evolution of two mutations. The Ile1,016/Phe1,534 haplotype appears to have low fitness suggesting that Ile1,016 was unlikely to have evolved independently. Instead the Cys1,534 mutation evolved first but conferred only a low level of resistance. Ile1,016 in S6 of domain II then arose from the Val1,016/Cys1,534 haplotype and was rapidly selected because double mutants confer higher pyrethroid resistance. This pattern suggests that knowledge of the frequencies of mutations in both S6 in domains II and III are important to predict the potential of a population to evolve kdr. Susceptible populations with high Val1,016/Cys1,534 frequencies are at high risk for kdr evolution, whereas susceptible populations without either mutation are less likely to evolve high levels of kdr, at least over a 10 year period.Pyrethroid resistance in the  Aedes aegypti, the primary mosquito vector of dengue, yellow Fever, and chikungunya viruses, pyrethroid resistance is primarily associated with mutations in the voltage-gated sodium channel protein. One mutation occurs in codon 1,016 and involves a replacement of valine with isoleucine , and a second located in subunit 6 of domain III in codon 1,534, replaces phenylalanine with cysteine . In Mexico, we found that Cys1,534 was present in the same mosquito collections that were previously analyzed for Ile1,016. In this study, we performed a linkage disequilibrium analysis on both Ile1,016 and Cys1,534 in Mexican collections from 2000–2012. Our analysis suggests that pyrethroid resistance requires the sequential evolution of the two mutations and that Cys1,534 must occur first and appears to enable the Ile1,016 mutation to survive.Constant use of pyrethroid insecticides has driven mosquito populations to develop resistance. In  Aedes aegypti (L.) mosquitoes are the principal urban vectors of dengue, chikungunya, and yellow fever viruses. Approximately 2.5 billion people (40% of the human population) currently live with the risk of dengue transmission. In Mexico, Ae. aegypti is the primary vector of the four dengue virus serotypes (DENV1-4), the causative agents of dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Mexico is severely affected by DF, DSS, and DHF because all four dengue serotypes co-occur in most states of Mexico. A recent review of dengue disease in Mexico [Worldwide Ae. aegypti is through reduction of larval and adult populations. In Mexico larval reduction is accomplished chiefly through the application of the organophosphate temephos to peridomestic larval breeding sites and through physical source reduction or alteration of potential water-holding containers. Following recommendations of the official Mexican policy for vector control, (NOM-032-SSA2-2002), pyrethroids were almost exclusively used to control adults in and around homes from 1999 to 2010.Currently the most effective means to reduce dengue transmission by Pyrethroid insecticides prolong the opening of the voltage gated sodium channel protein (VGSC) in insect nerves to produce instant paralysis and ‘‘knock-down.” The α-subunit of VGSC has four repeat domains, labeled I-IV, each of which contains six transmembrane helix segments, S1-S6. Pyrethroids preferentially bind to the open state of vgsc by interacting with two distinct receptor sites formed by the interfaces of the transmembrane helix S6 of domains II and III, respectively  suggest Ae. aegypti that is associated with very high knock-down resistance (kdr) to the pyrethroid insecticide permethrin in mosquitoes homozygous for this mutation. We examined collections of Ae. aegypti from Mexico during 1996–2009 [vgsc mutation was described in the S6 of domain III in Ae. aegypti that was also strongly correlated with kdr and involved a cysteine replacement  [In 2006 we described a mutation, Ile1,016, in the S6 of domain II in 996–2009  and founent Cys1,34Phe [5–,534Phe) . A generAe. aegypti for Ile1,016 and Cys1,534 while DNA previously analyzed for Ile1,016 [Based upon these observations and on the dual binding model , we analIle1,016 . An analLarval mosquitoes were collected from the locations mapped in 3 generation of the Viva Caucel and Vergel strains were exposed to 25 μg permethrin  coated 250 mL Wheaton bottles. In each bottle approximately fifty 3–4 days old mosquitoes were exposed for one hour. Active mosquitoes were transferred to cardboard cups and frozen at -80°C and formed the ‘alive’ group. Knocked down mosquitoes were transferred to a second cardboard cup and placed into an incubator at 28°C and 70% humidity. After four hours, newly recovered mosquitoes were aspirated, frozen and labeled as ‘recovered’. The mosquitoes that remained inactive were scored as ‘dead’.The Fij) that an allele at locus i = 1,016 appears with an allele at locus j = 1,534 was determined by the program LINKDIS [ij [There are four potential 1,016/1,534 dilocus haplotypes: Val1,016/Phe1,534 (VF), Val1,016/Cys1,534 (VC), Ile1,016/Phe1,534 (IF), Ile1,016/Cys1,534 (IC). The number of times  around pi and pj were calculated in WinBUGS[Where N is the sample size and 2 test was performed to determine if significant disequilibrium exists among all alleles at 1,016 and 1,534. The statistic was calculated and summed over all two-allele-interactions [A χractions :χ[1d.f.Rij [trans) to 0 , to 1 (both mutations cis) and therefore provides a standardized measure of disequilibrium:The linkage disequilibrium correlation coefficient Rij  is distrCi term corrects for departures from Hardy-Weinberg expectations:Hobs(i) is the observed frequency of i homozygotes. Departures from Hardy-Weinberg expectations were also expressed as Wright’s inbreeding coefficient (FIS) and calculated as 1- (Hexp/2p (1- p)) where Hexp is the observed frequency of heterozygotes. A χ2 test of the hypothesis FIS = 0 with one degree of freedom is:Where the The locations of all sampling sites are shown in In Viva Caucel, the frequency of the Ile1,106 allele was 0.746 and the frequency of the Cys1,534 allele was 0.926 , while iGenotypes at the 1,016 and 1,534 loci were not independent, in agreement with the linkage disequilibrium analysis in However, a problem with this analysis is that genotypes at the two loci are not independent. In this and previous studies , 11, Ile2 = 7.59, df = 1, p-value = 0.006).In general, numbers of recovered in the Viva Caucel strain were heavily dependent on genotypes at both loci. An interesting difference between the two loci is that 32% (28/88) of Ile1,016 heterozygotes recovered while only 3.6% (1/28) of Cys1,534 heterozygotes recovered. This difference was significant (χIS was significantly greater than zero (heterozygote deficiency) in two of the 36 collections where Ile1,016 and Val1,016 alleles were segregating. In contrast, a significant heterozygote deficiency occurred in eight of the 53 collections where Cys1,534 and Phe1,534 were segregating and an heterozygote excess occurred in two collections.Ae. aegypti collection sites, 80 out of 87 sites  of datasets. For the most part, alleles in 1,534 and 1,016 were evolving in a correlated, dependent fashion. However, this analysis does not provide specific information about the four haplotypes.Linkage disequilibrium analysis can only be performed in datasets where alleles are segregating at both loci. There were 34 datasets that met this criteria of the 87 collections listed in The frequencies of the four potential dilocus haplotypes are plotted by year in Ae. aegypti collected in Mexico from 2000–2012 , which infers strong positive selection acting on a recent mutant [An. gambiae, Tyr1,575 appears to have evolved on a Phe1,014 background. There are many potential reasons for this difference including the possibility that mutations within the S6 of domain III may produce a different resistance mechanism and have a different impact on fitness than mutations in the linker between domains III and IV. It is also possible that the specific changes of amino acids at these sites are unique and may confer different resistance phenotypes. In either case it seems likely that one of the mutations compensates for deleterious fitness effects of the other mutation and/or confers additional resistance to insecticides.Study in which t mutant . In contAn interesting difference between the two mutations in the present study is that 32% of Ile1,016 heterozygotes recover from pyrethroid exposure but only 3.6% of Cys1,534 heterozygotes recover. Thus while Cys1,534 in synergy with Ile1,016 may confer greater survival following pyrethroid exposure, Ile1,016 may confer a greater ability to recover following knockdown in heterozygotes.IS among these eight collections was large and positive (0.580) while the average among all collections was 0.052. This suggests that the fitness of Phe1,534 and Cys1,534 homozygotes may be greater than the fitness of G/T heterozygotes (i.e. underdominance). While these parameters have been estimated at the 1,016 locus [There was evidence of heterozygote deficiency in eight of the 53 collections and the average F16 locus , no simiAe. aegypti vgsc that may affect pyrethroid resistance. Codon 989 in the “super-kdr” region of domain II was assessed and no mutations were found [Ae. aegypti vgsc are also poorly understood. Analysis of segregation between alleles at the 1,011 and 1,016 codons in F3 showed a high rate of recombination even though the two codons are only separated by a approximately 250 bp intron [Little is known of other mutations in the re found . Ile, Mere found  but thesre found . The recvgsc [An. gambiae was introduced alone into an Ae.aegypti sodium channel (AaNav1-1) [Xenopus oocytes [Recent work on the dual binding model may shed some light on the next steps in the evolution of pyrethroid resistance in the vgsc . The TyraNav1-1)  and then oocytes . Tyr1,57An. gambiae increased resistance to permethrin and deltamethrin by 9.8- and 3.4-fold, respectively [The rapid increase in Cys1,534  cannot bcis by crossing over. Model 2 instead proposes that 1,534 mutations occurred first because 1,016 mutations confer low fitness. Ile1,016 mutations then arose on a Val1,016/Cys1,534 background. These results suggest that knowledge of the frequencies of both 1,534 and 1,016 mutations are important to predict the potential of a population to evolve kdr. Obviously, the frequency of Ile1,016 by itself is a poor predictor (kdr evolution. If our experience in tracking the frequencies of Ile 1,016 and Cys1,534 mutations over the past 15 years can be extended to other Ae. aegypti populations, then populations with intermediate to high frequencies of Cys1,534 might only be susceptible for 5–10 years. Conversely, pyrethroid susceptible populations without either mutation are unlikely to develop kdr quickly and might be susceptible for up to 10–15 years.redictor . Populat


Yarrowia lipolytica has emerged as a paradigm organism for the production of bio-lipids in white biotechnology. It accumulates high amounts of lipids from glucose as sole carbon sources. Nonetheless, to lower the cost of microbial oil production and rival plant-based fuels, the use of raw and waste materials as fermentation substrate is required. Starch is one of the most abundant carbohydrates in nature and it is constituted by glucose monomers. Y. lipolytica lacks the capacity to breakdown this polymer and thus expensive enzymatic and/or physical pre-treatments are needed.In the last year, the worldwide concern about the abuse of fossil fuels and the seeking for alternatives sources to produce energy have found microbial oils has potential candidates for diesel substitutes. Y. lipolytica. The modified strains were able to produce and secrete high amounts of active form of both proteins in the culture media. These strains were able to grow on starch as sole carbon source and produce certain amount of lipids. Thereafter, we expressed both enzymes in an engineered strain able to overaccumulate lipids. This strain was able to produce up to 21 % of DCW as fatty acids from soluble starch, 5.7 times more than the modified strain in the wild-type background. Media optimization to increase the C/N ratio to 90 increased total lipid content up to 27 % of DCW. We also tested these strains in industrial raw starch as a proof of concept of the feasibility of the consolidated bioprocess. Lipid production from raw starch was further enhanced by the expression of a second copy of each enzyme. Finally, we determined in silico that the properties of a biodiesel produced by this strain from raw starch would fit the established standards.In this work, we express heterologous alpha-amylase and glucoamylase enzymes in In this work, we performed a strain engineering approach to obtain a consolidated bioprocess to directly produce biolipids from raw starch. Additionally, we proved that lipid production from starch can be enhanced by both metabolic engineering and culture condition optimization, setting up the basis for further studies.The online version of this article (doi:10.1186/s13068-015-0335-7) contains supplementary material, which is available to authorized users. The excessive use of petroleum sources cannot cope with the increasing worldwide energy consumption and the environmental requirements to prevent global warming. Bio-lipids produced either by plant or microorganisms are one of the most promising sustainable and renewable alternative sources of energy . Microbid-glucopyranose chains with α-1,6 branching points) and amylose chain helix . It is an import food and feed source and a preferred substrate for the production of many industrial products. In fact, the use of starch to produce biofuels, such as bioethanol, is already a consolidated technology [Saccharomyces cerevisiae into amylolytic yeast. Therefore, many different amylases have been expressed in baker yeast to make it able to produce ethanol from starch in CBP manner [Starch is a polymer of glucose formed by amylopectin   Fig.  or in rad-glucose units and saccharification of the polymers [A. niger [S. cerevisiae using starch as sole carbon source are based in the single heterologous expression of glucoamylase [Aspergillus was essential in the saccharification step prior to a fermentation to produce lipids using Lipomyces starkeyi [Y. lipolytica wild-type strain (Po1d) generating the strain JMY5083. Glucoamylase signal sequence was replaced by the targeting sequence of Lip2 and its expression was controlled by the promoter TEF  Fig.  and in mNonetheless, this strain was unable to grow in raw starch medium (RS). This result may be explained due to a lack of α-amylase activity Fig.  which maY. lipolytica expressing α-amylase is able to clarify raw starch and how the strain expressing glucoamylase can release enough glucose from soluble starch to permit growth. Nonetheless, the single expression of one of the amylases is not enough to make Yarrowia able to use raw starch as carbon source. Therefore, we decided to combine the two enzymes in the same strain. In addition, synergistic effects have been found in the co-expression of both enzymes as well as higher titers in biomass and ethanol production from starch in baker yeast [We showed that er yeast –39. As eC/N ratio, which is known to trigger lipid accumulation in this yeast [C/N ratio of 60. When the C/N ratio was increased to 90, the same strain doubles its lipid accumulation capacity, reaching 7.2 ± 0.4 % of the DCW as fatty acids . We therefore make cultures in two different media with high is yeast . The modids Fig. a, b. In ids Fig. c, d as iY. lipolytica containing multiple modifications able to accumulate high amounts of lipids, JMY3501 [POX genes, avoiding lipid degradation in the peroxisome [TGL4, the main intracellular lipase in charge of the release of fatty acids from TAGs stored in the lipid body [GPD1 [DGA2 [We have previously generated an engineered strain of  JMY3501 . This stroxisome . It has pid body . Additiody [GPD1  involvedD1 [DGA2 , encodinC/N ratios (60 and 90). As we expected, JMY5035 strain greatly improved lipid content, up to 21.1 ± 1.4 % of the DCW, 5.7 times more than JMY5017  presenting different N ratios 0 and 90.N ratios 0 and 90.ty acids . SimilarY. lipolytica strains can accumulate lipids from starch but also that the amount of produced lipids can be enhanced by both optimization of culture conditions and strain engineering.Therefore, we here not only proved that our modified Y. lipolytica as a consolidated bioprocess to produce lipids from starch, we wanted to test our strains in an industrial starch media (IS). For this purpose, we obtained starch from the company Tereos Syral and we used it directly as a substrate for flask fermentation. To cope with the higher amounts of substrates normally used in large-scale fermentations, we constructed additional strains expressing one extra copy of each of the enzymes (JMY5196) using JMY5035 as parental strain. These strains were able to grow and produce lipids using the industrial product containing starch as carbon source (IS media) in flask under non-optimized conditions . The parental strain JMY3501 produced similar lipid titers, 3.2 g/L from 60 g/L of glucose, and therefore with a lower yield YFA/S = 0.053 g/g [In addition, we proved that a second copy of both α-amylase and glucoamylase further increase lipid production up to 2.84 ± 0.16 g/L, which represent a yield .053 g/g . Althoug.053 g/g , here weY. lipolytica to bind plant biomass, which could be useful in cell immobilization approaches. Additionally, this interesting feature could be exploited towards the use of lignocellulosic material using this yeast. This fact is in accordance to previous reports that show the ability of Yarrowia to bind to hydrophobic substrates in an inducible manner [Surprisingly, we found those strains able to grow in the industrial starch on the surface of the insoluble fractions (coming from wheat cells) present in this substrate, as revealed in the electronic microscopy [Fig. e manner  due to Le manner  NonethelY. lipolytica biolipids for biodiesel production were analyzed according to the most restrictive standard, EN 14214. We therefore evaluated the following parameters  . It is iY. lipolytica, α-amylase from O. sativa and glucoamylase from A. niger. To our knowledge this is the first report on the expression of an active glucoamylase in Yarrowia. The modified strain was able to grow in starch as sole carbon source. Additionally, we performed the same expressions in a lipid accumulating background and it proves to accumulate high amounts of lipids. Interestingly, the amount of intracellular lipid was not only dependent in the genetic background but also in the C/N ratio of the media, which proof the potential enhancement of the yield and productivity of the process after culture optimization at bioreactor scale. Finally, the engineered strain was able to produce lipids from industrial raw starch what is a proof of concept in the use of Y. lipolytica as a consolidated bioprocess to generate biodiesel. Interestingly, the theoretical analysis of the biodiesel properties of the FAMEs generated from starch fits the most restrictive regulatory standard.In summary, we successfully expressed and secreted heterologous amylases in Y. lipolytica strains used in this study were derived from the wild-type Y. lipolytica W29 (ATCC20460) strain. All of the strains used in this study are listed in Table Escherichia coli and Y. lipolytica have been described elsewhere [4Cl and 50 mM phosphate buffer (pH 6.8). Soluble starch medium (SS) was similar to YNBCN60 but substituting glucose for soluble starch, 1 % in the case of microplate assay (see results), 6 % in C/N60 and 9 % in C/N90, all of them contained 0.17 % (wt/vol) yeast nitrogen base (YNBww), 0.15 % (wt/vol) NH4Cl and 50 mM phosphate buffer (pH 6.8). Raw starch medium (RS) was similar to YNBCN60 by substituting glucose for wheat raw starch (1 %) (Sigma-Aldrich), and as YNBCN60, 0.17 % (wt/vol) yeast nitrogen base (YNBww), 0.15 % (wt/vol) NH4Cl and 50 mM phosphate buffer (pH 6.8). The industrial starch medium (IS) was similar to YNBCN60 but substituting glucose for 25 % industrial product containing starch (DZ starch) provided by Tereos Syral (Belgium).The lsewhere . MinimalY. lipolytica (GenScript) . PCR amplifications were performed in an Eppendorf 2720 thermal cycler with GoTaq DNA polymerases (Promega). PCR fragments were purified with QIAgen Purification Kit . All the reactions were performed using the manufacturer instructions.Supernatant of 6 days culture in YNB were concentrated 10× using Amicon Ultra-0.5 10 K centrifugal filters device . The concentrated supernatant was loaded into a 10 % SDS-PAGE gel to see the expression and secretion of the heterologous proteins. The molecular weight marker Mark12™ unstained was used as standard (Novex).Starch-containing plate of YPD was used to determine α-amylase activity. After 3 days of either growing cells or incubating with 5 μl drop from culture supernatant at 28 °C, the plate was stained with iodine vapor. The clear zones around the colonies or the supernatant drops determined the presence of α-amylase activity.To determine DCW in flask experiment, 2 mL of the culture was washed and lyophilized in a pre-weighted tube. The differences in weight correspond to the mg of cells found in 2 mL of culture.600 nm) at different intervals, with a microtiter plate reader . For each strain and set of conditions, we used two biological replicates.Growth tests were performed in 100 µL cultures in 96-well plates, with constant shaking, in the presence of 1 % soluble starch as the carbon source. Precultures were grown on minimal medium plates, as for the growth tests. Growth was monitored by measuring the optical density  using an Aminex HPX87H column coupled to UV (210 nm) and RI detectors. The column was eluted with 0.01 N HLipids from aliquots of 10–20 mg of cells were converted into their methyl esters with freeze-dried cells according to Browse et al.  and used® Lipid Probe  to the cell suspension (A600 of 5) and after incubation for 10 min at room temperature.Images were acquired using a Zeiss Axio Imager M2 microscope  with a 100× objective and Zeiss filters 45 and 46 for fluorescent microscopy. Axiovision 4.8 software  was used for image acquisition. Lipid bodies visualization was performed by addition of BodiPyBacillus subtilis biofilms was performed at an operating voltage of 2 kV under an S-4500 Hitachi FESEM  at the MIMA2 platform.Electronic microscopy was performed as INRA-AgroParisTech platform Microscopie et Imagerie des Micro-organismes, Animaux et Aliments . The samples were critical point dehydrated  using carbon dioxide as the transition fluid and coated with gold–palladium (272 Ǻ of thickness) in an automatic sputter . High-magnification imaging of the Y. lipolytica engineered strains grown in industrial starch. The equations and models used have been previously described in detail by Ledesma-Amaro et al. [Mathematical equations and predictive models were used to theoretically determine all the physicochemical fuel-related parameters of the biodiesel obtained by transmethylation of o et al.  based ono et al. , 57–59.


Background: Several monoclonal antibodies (mAb) are being evaluated as treatment options for the current 2014 Ebola outbreak. But they were derived from and tested for protection against the older 1976 Mayinga or 1995 Kikwit Zaire Ebolaviruses (EBOV). The EBOV sequences reported for the current outbreak contain several mutations whose significance remained to be established.Methods: We analyzed sequence and structural conservation of the Ebolavirus glycoprotein (GP) epitopes for all experimentally identified protective mAbs published to date.Results: The conservancy analysis of protective mAb epitopes in the Ebolavirus glycoprotein sequences spanning all Ebola virus lineages since 1976 showed that conservancy within the Zaire EBOV lineage was high, with only one immunodominant epitope of mAb 13F6-1-2 acquiring two novel mutations in the 2014 outbreak that might potentially change the antibody specificity and neutralization activity. However, the conservation to other Ebola viruses was unexpectedly low.Conclusion: Low conservancy of Zaire EBOV mAb epitopes to other EBOV lineages suggests that these epitopes are not indispensable for viral fitness, and that alternative mAbs could be developed to broadly target all EBOV. However, average percent sequence identity of the epitopes for mAbs used in current cocktails to the Zaire EBOV is high with only one epitope differing in the 2014 outbreak. These data bode well for general usefulness of these antibodies in the context of the current outbreak. Sequences were aligned using the MAFFT L-INS-IThe epitopes were mapped on the GP sequence based on epitope residue positions provided in the IEDB; the specific strain provided for each epitope in the IEDB allows for unambiguous residue mapping.Residue conservation was calculated for the GP protein, using the ConSurf2QHR] after energy minimization.FoldXin vitro correlates of protection  or in vivo survival assays, so-called ‘functional epitopes,’ revealed ten epitopes for nine mAbs . Four of these epitopes were discontinuous, with two, 16F6 and KZ52, obtained from the X-ray structures of the GP-antibody complexes, and the other two, 133/3.16 and 226/8.1, from the experiments using escape mutants. Only one of the nine mAbs was derived from a human survivor of the Ebola hemorrhagic fever (EHF) of the 1995 Kikwit outbreak (mAb KZ52). All other mAbs were either obtained from a murine model or an engineered Fab derived from a display library.As of September, 2014, analysis of the Ebola GP protein epitopes reported in the Immune Epitope Database (IEDB) and identified using assays demonstrating Figure 2. Three of these epitopes, 6D8, 13F6, and 4G7, are mapped to the heavily glycosylated mucin-like domain of the Ebola GP protein spanning the GP1 residues 313 to 501. This domain is unique to GP, includes the GP surface available for host interaction, and is not shared with sGP , thus making it a desirable target for the Ebola immunotherapeutics3CSY, 3VE0, 3S88], due to its increased flexibilityThe mapping of the functional mAb epitopes to the alignment of the 42 non-identical sequences of the GP proteins from all EBOV is shown in Figure 3. It can be seen that conservancy in other than Zaire EBOV – that is, Bundibugyo, Tai Forest, Reston, and Sudan viruses – varied from 0 to 90%, with the 2G4 epitope being the most conservative (above 78%) and the 6D8 and 13F6 epitopes, the least conservative (below 23%). For the Zaire EBOV, average sequence identity of all but one epitope was above 98% in both previous and current outbreaks.The sequence conservancy of six of the functional epitopes for the most tested protective mAb cocktails, ZMab, ZMapp, and MB-003, were evaluated for all Ebola viruses and is provided in This epitope is the immunodominant epitope 404-VEQHHRRTDND-414 of the GP1 protein recognized by mAb 13F6-1-2 (part of mAb cocktail MB-003). It was identical in all Zaire EBOV since 1976 and was considered a good target for vaccine immunotherapeutic design, as it is immunodominant in a murine model, located in the mucine-like domain, provides protection against viral challenge, and does not adopt any secondary structure or posttranslational modifications that would be difficult to achieve in synthetic or recombinant systems2QHR] shows that while the E405G mutation would unlikely have an effect on the antibody binding, the T411A mutation would disrupt a tight hydrogen bond (2.7 Å) between the Thr411 Oγ side-chain atom of the peptide to the Asp33 Oδ1 side-chain atom of the antibody heavy chain, potentially changing the antibody specificity and neutralization activity. Indeed, estimating the effect of this mutation in the 2QHR structure with FoldX predicts the destabilization of the peptide-antibody interaction by 1.24 kcal/mol .However, this epitope acquired two novel mutations, namely E405G and T411A that are present in all sequences of the 2014 outbreak. The crystal structure of mAb 13F6-1-2 in complex with this peptidic epitope3CSY] one can visualize the distribution of conserved and variable regions on the surface . Further considering escape mutants and antibody complexes with the GP trimer , suggests a consensus binding region comprised by the conformational epitope  where mAb 2G4 (part of ZMab and ZMapp) is expected to bindBy mapping the sequence conservation from the non-redundant EBOV alignment to the GP trimer structure PDB: CSY one cThe presented analysis of mAb epitopes is novel in respect to a complete conservancy analysis spanning all Ebola virus lineages since 1976 until now and considers the GP protein not only at the sequence but also the structural level as several of the epitopes are conformational. Our two main findings from the analysis are the following: (1) among 6 epitopes targeted by different mAb cocktails only one seems to be significantly different in the current outbreak compared to the earlier ones; and (2) the conformational epitope of GP2 targeted by multiple mAbs includes a region that is not well conserved among the different lineages, indicating that cross-protection from non-Zaire outbreaks to the current one may be limited.The provided sequence and structural mapping of the epitopes also allows closely following the further evolution of these sites within the outbreak as new sequences become available and hence should be a useful contribution to the current joint global efforts to tackle the serious outbreak.


Starch and proteins are major components in the wheat endosperm that affect its end product quality. Between the two textural classes of wheat i.e. hard and soft, starch granules are loosely bound with the lipids and proteins in soft wheat due to higher expression of interfering grain softness proteins. It might have impact on starch granules properties. In this work for the first time the physiochemical and structural properties of different sized starch granules  were studied to understand the differences in starches with respect to soft and hard wheat. A-, B- and C-type granules were separated with >95% purity. Average number and proportion of A-, B-, and C-type granules was 18%, 56%, 26% and 76%, 19%, 5% respectively. All had symmetrical birefringence pattern with varied intensity. All displayed typical A-type crystallites. A-type granules also showed V-type crystallinity that is indicative of starch complexes with lipids and proteins. Granules differing in gelatinization temperature (ΔH) and transition temperature (ΔT), showed different enthalpy changes during heating. Substitution analysis indicated differences in relative substitution pattern of different starch granules. Birefringence, percentage crystallinity, transmittance, gelatinization enthalpy and substitution decreased in order of A>B>C being higher in hard wheat than soft wheat. Amylose content decreased in order of A>B>C being higher in soft wheat than hard wheat. Reconstitution experiment showed that starch properties could be manipulated by changing the composition of starch granules. Addition of A-granules to total starch significantly affected its thermal properties. Effect of A-granule addition was higher than B- and C-granules. Transmittance of the starch granules paste showed that starch granules of hard wheat formed clear paste. These results suggested that in addition to differences in protein concentration, hard and soft wheat lines have differences in starch composition also. Wheat is one of the most important cereals for direct human consumption. Its textural properties determine the end product quality. Wheat has two textural classes i.e. soft and hard. Two classes of wheat are utilized for different purposes. Hard wheat is mainly used for bread making, pasta making while soft wheat is utilized for biscuit and cake making. The differences in grain hardness arise due to presence or absence of two puroindoline proteins PINA and PINB . The preTriticum aestivum cv. C306) obtained from Directorate of Wheat Research, Karnal, India  and a land race (T. aestivum L. IITR67) obtained from Prof. H.S. Dhaliwal, Indian Institute of Technology, Roorkee and collected from Hilly region of Uttarakhand, India . These two genotypes with contrasting hardness were grown in the experimental field of National Agri-Food Biotechnology Institute (NABI), SAS Nagar  Punjab, India . The harvested seed at maturity were used for starch isolation.Two wheat genotype, a Indian wheat cultivar .Wheat starch granules were isolated following the method of Peng et al. . BrieflySedimentation was used to separate total starch into three fractions A-, B- and C-granules as described by Takeda et al.  with modThe size of starch granules i.e. A-, B-, C-granules and total starch (T-type) granules were analyzed by light microscopy. Starch granules (4 mg) were suspended in 200 μl of 90% ethanol. A drop of starch suspension was spread on a microscope slide and air-dried. The slide was then placed on the stage of a light microscope  and analyzed for granule size, shape and birefringence (using polarized light mode). Number of different type of starch granules from total starch was counted using microscopic counter  after staining with potassium iodide (KI).Scanning electron microscopy  was used to assess the shape, size and purity of different starch granules. Starch samples (~1 mg) were applied on an aluminum stub using double-sided adhesive tape, and the starch was coated with gold. An acceleration potential of 10.0 kV was used during microscopy at 30X.10 mg of starch sample was dissolved in 2 ml of DMSO in a boiling water bath. After passing through the Sepharose CL-2B column , amylose and amylopectin were separated based on their molecular weight. Fractions were assayed using phenol-sulphuric acid method . The chrXRD of starch granules  were recorded using diffractometer , Cu KR radiation with a wavelength of 0.154 nm operating at 45 kV and 40 mA. XRD were acquired at 25°C over a 2θ range of 3–40° with a step size of 0.018° and sampling interval of 52 s. Relative crystallinity was estimated from the ratio of the area of peaks to the total area of diffractogram (2θ).Thermal properties were analyzed using DSC  equipped with a thermal analysis data station. The analyzer was calibrated using indium and an empty aluminum pan was used as reference. Sample pans (5 mg starch in 15 μl of water) were heated at a rate of 5°C/min from 40 to 110°C and cooled at the same rate to 40°C. The heating and cooling cycles were repeated three times. Onset temperature (To), peak temperature (Tp), conclusion temperature (Tc) and enthalpy (ΔH) were calculated using stare software for thermal analysis (STARe SW 9.01).Starch granules mixtures were prepared by individually adding 10% (w/w) of A-, B- and C-granules extracted from soft wheat to soft wheat total starch. Similarly, isolated granules of hard wheat were added to hard wheat total starch. In addition, hard wheat granules were added to soft wheat and vice versa. Thermal properties were analyzed using DSC.e5, Molecular Devices, Sunnyvale, California, US).Starch granules paste (1%) was prepared by dissolving 10 mg of starch granules in 1 ml of water in boiling water bath for 30 min. After cooling for 5 min, percent transmittance (%T) was determined against water blank at 650 nm using spectrophotometer  by Duncan’s test (p< 0.05) using SPSS 16.0 software .In the present work we were able to separate A-, B- and C-type starch granules with >95% purity. Previous studies have reported morphological data of A- and B-type starch granules ,20. In tLight and scanning electron microscopy of total starch, A-, B-, C-granules from soft and hard wheat indicated significant size variation between different types of granules and non-significant variation within starch granules from different textured wheat . A-granuThe intensity of birefringence depends on the granule size, relative crystallinity, and microcrystalline orientation. Polarized microscopy of A-, B- and C-granules indicated that A-granules had higher birefringence intensity than B- and C-type granules . RelativX-ray diffraction pattern of total starch, A-, B- and C-granules have been shown in Starch granule composition was calculated from elution peaks of gel permeation chromatography . Area unTransmittance of the starch granules  paste afThermal properties of the different starch granules as determined by DSC indicated that gelatinization enthalpy (ΔH) of different granules decreased in the order of A>B>C. ΔH has been related to degree of crystallinity . This reReconstitution of soft wheat total starch with 10% of its own individual A-, B- and C- granules indicated significant changes in ΔH  and ΔT . HighestCorrelation analysis of crystallinity with amylose content has been shown in Isolated amylose molecules showed crystalline structure with digestion resistance . DigestiSubstitution analysis of different starch granules as carried out by GC/MS. Differences were observed in structure and physiochemical properties between A-, B- and C-type granules isolated from hard and soft wheat. A-granules from hard wheat showed higher degree of substitution, crystallinity, enthalpy and lower amylose content. C-granules from soft wheat showed low crystallinity and higher amylose content. This study can form the basis for further comprehensive characterization of starch granules from soft and hard wheat.S1 FigS, AS, BS and CS represents soft wheat total starch, A granules, B granules and C granules respectively. TH, AH, BH and CH represents hard wheat total starch, A granules, B granules and C granules respectively.T(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIFF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


Correction to:British Journal of Cancer (2015) 114, 146–150; doi:10.1038/bjc.2015.421; published online 15 December 2015British Journal of Cancer, one of the authors identified an error in the spelling of their name. V Sivasubramaniam appears correctly in the author list above.Upon publication of the above paper in the


This does not alter adherence to all the PLOS ONE policies on sharing data and materials.The following information is missing from the Competing Interests section: The authors have read the journal's policy and have the following conflicts: M.L., D.S. and G.G. are inventors of the polyfunctionality index (patent number: WO2013127904) and M.L. is proprietary owner of the Funky Cells ToolBox software (


In this period, I could not start this editorial without wishing all of you a Happy New Year.This is also the opportunity for me to warmly thank, for their confidence, our authors, readers, reviewers, members of the editorial board, sponsors, as well as members of MDPI AG in Basel, Beijing, and Wuhan.Pharmaceuticals at the end of January, I was told that the job would be challenging and exciting. It was true, but I got a great deal of support from the scientific and administrative teams of MDPI AG. That allowed us to accumulate excellent results during the past year. Visibility of Pharmaceuticals increased from 458,383 full-text views in 2014 to 608,080 in 2015, a jump of almost 30%. Three Special Issues have been successfully completed. They were dedicated to mitochondrial target-based drug discovery [When I was offered the position of Editor-in-Chief of In 2015, eight distinguished scientists joined the Editorial Board: Jean Pierre Bazureau , Tien L. Huang , Joachim Jose , Christopher W. K. Lam , Viktor P. Lozitsky , Louis M. Mansky , Suzanne Peyrottes , and Maria Emilia de Sousa . Annie Mayence  acts as a new associate editor.Pharmaceuticals has affirmed its status of international scientific journal by sponsoring a series of events among which:23. Jahrestagung der Arbeitsgemeinschaft Radiochemie/Radiopharmazie, Erlangen, Germany;èmes Journées Franco-Belges de Pharmacochimie, Spa, Belgium;291st joint European Conference on Therapeutic Targets and Medicinal Chemistry, Münster, Germany;23rd National Meeting on Medicinal Chemistry, Salerno, Italy;11° Encontro Nacional de Quimica Organica/4° Encontro de Quimica Terapeutica, Porto, Portugal;Ricai 2015: 35th Interdisciplinary Meeting of Anti-infective Chemotherapy, Paris, France.Pharmaceuticals was the proud organizer of the First International Electronic Conference of Medicinal Chemistry [The journal was also present at the 250th American Chemical Society National Meeting and Exhibition in Boston, MA, USA. In addition, last but not least, hemistry . Membershemistry .Pharmaceuticals. We are also proud to let you know that the journal will organize a competition awarding one travel grant [No time for napping; in 2016 we must confirm the promising results obtained during 2015. Increasing the visibility of our journal will remain one of our main goals. We are already working in that direction, and we are pleased to announce that the Second International Electronic Conference on Medicinal Chemistry  will takel grant . It will


Endothelial keratoplasty (EK) is used increasingly for the management of cases with endothelial dysfunction. Long-term outcomes of the surgery are not widely reported in the literature. We report our experience of EK in Chinese eyes at a University teaching hospital.Retrospective analysis was performed for all cases of EK performed between 2005 and 2009. Data analyzed included indication for surgery, associated surgical procedures, complications, best-corrected visual acuity (BCVA) and overall graft survival.n = 12) followed by Fuchs’ endothelial dystrophy (n = 4), or both (n = 3). Other indications for surgery included failed penetrating keratoplasty (n = 2) and endothelial decompensation due to multiple surgeries (n = 1). Triple procedure was performed in 5 (22.7%) cases. Complications were noted in the form of postoperative interface hemorrhage  and graft dislocation . The mean postoperative endothelial cell density was 1069 ± 585.8 cells/mm2. The mean postoperative pachymetry was 675.8 ± 108.5 μm. The mean preoperative and postoperative intraocular pressure was 11.3 ± 3.2 and 13.9 ± 4.5 mmHg respectively. At the last follow-up , BCVA was ≥20/70 in 9 (40.9%) cases. Causes of poor BCVA included primary graft failure (n = 4), graft decompensation (n = 4), advanced glaucoma (n = 2) and irreversible graft rejection (n = 2). Graft remained clear in 12 (54.5%) cases at the last follow-up. Average graft survival was 19.7 ± 17.7 months (median 17.5 months).Overall, 22 eyes of 21 patients underwent EK . Pseudophakic bullous keratopathy was the leading indication for surgery (Nearly half of the EK grafts in our study survived over a period of five years. Graft failure, glaucoma and graft rejection were the main causes of poor graft survival. Endothelial keratoplasty (EK) has become the treatment of choice in cases with corneal endothelial dysfunction in the presence of clear corneal stroma. Over the past few years, the spectrum of indications of EK is expanding ,2]. It . It 2]. 2 were used in all cases. All surgeries were performed using a standard surgical technique as described previously . Al. Al21]. n = 4) and late graft failure (n = 4), and advanced glaucoma (n = 2). The reported [In our study, nearly half of the grafts were clear at the last follow-up. The main causes of poor graft survival were primary (reported  extrapolreported  a statisLimitations of the present study include a retrospective design and small sample size. Also, we do not have data on endothelial cell counts and corneal thickness after EK. Nevertheless, our study presents the long-term follow-up outcomes after EK in Chinese eyes. Similar studies from other centers would be useful in order to compare EK and penetrating keratoplasty over an extended follow-up period.


The following scientists assisted the journal by reviewing manuscripts during the period October 1, 2014 to September 30, 2015. We gratefully acknowledge their critical evaluation and assistance in selecting articles for publication.


Graft ischemia results in inhibition of Na+–K+ ATPase, inhibition of K+ ATP channels, drop of intracellular K+, and the absence of flow favors cell membrane depolarization (+ ATP channels would be associated with increased NADPH oxidase (NOX2) activity and increased production of reactive oxygen species (ROS)  . Decreaspro IL18 . Both IL++-sensitive K+ channels during lung graft ischemia would be expected to provide protection through antagonizing membrane depolarization , which would attenuate ROS production, leading to abortion of inflammasomes priming and activation, and accordingly the release of pro-inflammatory cytokines.Accordingly, the enhancement of Caex vivo lung perfusion , which correlated with decreased incidence of primary graft dysfunction and chronic lung allograft dysfunction after transplantation (++-activated K+ channels (The Toronto team of lung transplantation has achieved significant inhibition of cytokines production within the lung graft through gene therapy during antation . Howeverantation . HO-1 cachannels .++-activated K+ channels during lung graft ischemia. Accordingly, further studies should be conducted to investigate the actual status of these channels during lung graft ischemia prior to transplantation. In addition, pharmacological activation of these channels could be a good target to protect the lung graft during transplantation, with corresponding improvement of the clinical outcome.These findings highlight the possible protective role of the enhancement of CaThe author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Journal of Functional Biomaterials is instituting an annual award to recognize the outstanding papers related to materials for biomedical applications published in this journal. We are pleased to announce the first “Journal of Functional Biomaterials Best Paper Award” for 2015.The Journal of Functional Biomaterials based on the citations and downloads among all the research papers published in 2014 and 2013. We are pleased to announce that the following three papers were chosen by the Prize Awarding Committee:Best Paper Award1st PrizeVictoria Leszczak, Dominique A. Baskett and Ketul C. PopatSmooth Muscle Cell Functionality on Collagen Immobilized Polycaprolactone Nanowire SurfacesJ. Funct. Biomater.2014, 5(2), 58–77; doi:10.3390/jfb5020058http://www.mdpi.com/2079-4983/5/2/58Available online: A very comprehensive study of an important cell–material interaction that could be of wide interest to tissue engineers.”––Prof. Dr. Joachim B. Kohn“Advanced biomaterial concepts, comprehensive characterization.”––Prof. Dr. Aldo R. Boccaccini“A very accurate biological characterization.”––Dr. Antonella Sola“Useful study of SMC morphology and turnover on a surface of nanowires orientated vertically to the surface. Normally such structures are horizontally arrayed on the surface. More could have been done to rationalise the structure with a surface that has so many unusually deep vacant spaces. The physiological relevance of such an unusual surface is also not clear.”––Prof. Dr. Pankaj Vadgama“2nd PrizeAdel M.F. Alhalawani, Declan J. Curran, Belinda Pingguan-Murphy, Daniel Boyd and Mark R. Towlerex-Vivo StudyA Novel Glass Polyalkenoate Cement for Fixation and Stabilisation of the Ribcage, Post Sternotomy Surgery: An J. Funct. Biomater.2013, 4(4), 329–357; doi:10.3390/jfb4040329The article describes an interesting result but is only of limited interest to a specialized subgroup of scientists.”––Prof. Dr. Joachim B. Kohn“Comprehensive, scientifically sound, well written and completed by a rich bibliography.”––Dr. Antonella Sola“Exceptionally comprehensive study of based glass polyalkenoate cements with comparison of surface, bulk and ion release properties. It has breadth and sufficient bone fixation interest to be of significance to all who work in this field. However, it tends to veer towards the descriptive rather than a mechanistic analysis.”––Prof. Dr. Pankaj Vadgama“3rd PrizeDeepen Paul, Sharmistha Paul, Nima Roohpour, Mark Wilks and Pankaj VadgamaAntimicrobial, Mechanical and Thermal Studies of Silver Particle-Loaded PolyurethaneJ. Funct. Biomater.2013, 4(4), 358–375; doi:10.3390/jfb4040358http://www.mdpi.com/2079-4983/4/4/358Available online: The article describes an interesting result but is only of limited interest to a specialized subgroup of scientists. It’s a well done study.”––Prof. Dr. Joachim B. Kohn“Relevant, timely, well conducted study on Ag nanoparticles as part of a composite material.” ––Prof. Dr. Aldo R. Boccaccini“A step forward into the investigation of Ag use for antibacterial applications.”––Dr. Antonella Sola“Nominations were made by the Editorial Office of Journal of Functional Biomaterials. On behalf of the Prize Awarding Committee and the Editorial Board, we would like to congratulate these three teams for their excellent work. In recognition of their accomplishment, the corresponding authors of these three best papers, Dr. Ketul C. Popat, Dr. Mark R. Towler, Dr. Deepen Paul and Dr. Pankaj Vadgama will receive the privilege of publishing an additional paper free of charge in open access format in the Journal of Functional Biomaterials after regular peer-review procedures.These three outstanding papers are valuable contributions to the Prize Awarding CommitteeEditor-in-ChiefProf. Dr. Francesco PuociDipartimento di Scienze Farmaceutiche, Università della Calabria, Edificio Polifunzionale, Arcavacata di Rende (CS) 87036, Italyfrancesco.puoci@unical.itE-Mail: Associate EditorProf. Dr. Pankaj VadgamaInterdisciplinary Research Centre (IRC) in Biomedical Materials, Queen Mary, University of London, Mile End Road, London E1 4NS, UKp.vadgama@qmul.ac.ukE-Mail: Editorial Board MemberProf. Dr. Aldo R. BoccaccinInstitute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg, 91058 Erlangen, Germanyaldo.boccaccini@ww.uni-erlangen.deE-Mail: Editorial Board MemberProf. Dr. Joachim B. KohnThe New Jersey Center for Biomaterials, Rutgers, The State University of New Jersey, 145 Bevier Rd., Piscataway, NJ 08854, USAkohn@rutgers.eduE-Mail: Editorial Board MemberProf. Dr. Dusan LosicSchool of Chemical Engineering, The University of Adelaide, Adelaide, SA 5005, Australiadusan.losic@adelaide.edu.auE-Mail: Editorial Board MemberProf. Dr. Vladimir M. MirskyFaculty of Natural Sciences / Nanobiotechnology, Brandenburg University of Technology Cottbus-Senftenberg, 01968 Senftenberg, GermanyVladimir.Mirsky@HS-Lausitz.deE-Mail: Editorial Board MemberDr. Antonella SolaDepartment of Engineering “Enzo Ferrari”, University of Modena and Reggio Emilia, 41125 Modena, Italyantonella.sola@unimore.itE-Mail:


Langerhans cells (LCs) have been scrutinized many times in studies of the pathogenesis of graft-versus-host disease (GVHD). As migratory dendritic cells, LCs are capable of direct antigen presentation to cytotoxic T cells. Their self-renewal capacity has led to speculation that persistent recipient LCs could provide a continuous source of host antigen to donor T cells infused during hematopoietic stem cell transplantation (HSCT). In this issue of Experimental Dermatology, a new study examines at the relationship between recipient LCs and chronic GVHD. The role of Langerhans cells (LCs) in the pathogenesis of graft-versus-host disease (GVHD) remains enigmatic. LCs are the paradigmatic migratory dendritic cell (DC) and are potent at priming CD8+ T cells . In haemHuman studies had previously shown that LCs were in cell cycle  and undeExperimental Dermatology examines a new aspect, the relationship between persistent recipient LCs and the occurrence of chronic GVHD (A recent paper in nic GVHD . Perhapsin situ analysis and studies showing high donor chimerism in LCs isolated from epidermal sheets by migration (in vitro culture might somehow favour the detection of donor-derived LCs. However, there is no a priori reason to justify this, and early after transplantation, it is possible to recover high numbers of recipient LCs by migration.The surprising finding is not the lack of an association with chronic GVHD, which is postulated to depend more on indirect antigen presentation by donor-derived DCs –12, but igration ,8. Migrain situ methods are liable to overestimate the proportion of recipient LC nuclei because the intricate branching of LC membrane can lead to the inadvertent inclusion of recipient keratinocyte nuclei. This problem may be minimized using the more intense peri-nuclear distribution of Langerin to detect LC nuclei; Langerin also has the distinct advantage of protease resistance that permits immunofluorescence to be performed after FISH. In addition, Z-stack reconstructions ensure that scored nuclei are completely encapsulated in LC membrane, and post-GVHD skin may be used to provide controls in which a very high proportion of donor LCs is expected. Using these approaches, high donor LC engraftment was also recently observed in a study incorporating a large cohort of minimally conditioned patients (No technique is perfect, and patients .These considerations aside, Andani and colleagues have analysed a homogenous population of patients without GVHD, and their conclusion is that there is significant persistence of recipient LCs in the absence of skin inflammation. This is certainly in keeping with the reports of human limb transplantation, in which donor LCs survive long-term , and higGATA2 mutation lacking DCs but retaining LCs and macrophages also still experience GVHD (The role of LCs in murine transplantation models is still debated. While it is clear that LCs may trigger acute GVHD if they are the only recipient APCs remaining ,15, theynce GVHD –21.in vitro (A new facet of GVHD research is the potential of stable recipient macrophages to promote GVHD. In human transplantation, macrophages outlive LCs and can stimulate allogeneic memory T-cell responses in vitro . In micein vitro –25.In conclusion, we know that many recipient LCs are intact at the moment that donor T cells are infused into humans, and it remains difficult to rule out a contribution of recipient LC-mediated antigen presentation to acute GVHD. However, despite the persistence of many investigators, it remains increasingly hard to envisage that measurements of post-transplant LC chimerism will lead to useful clinical decisions.


We constructed a composite straight graft by combining a right internal thoracic artery and a radial artery. All the radial artery grafts were sequentially anastomosed to coronary arteries. We measured the diameters of the radial arteries before the operation, within 1 month and 1 year after the operation. At early postoperative period, graft diameter was significantly larger in skeletonized grafts. Graft diameter at the point before the first and the second anastomosis was similar in skeletonized grafts, although that was significantly smaller before the second anastomosis in non-skeletonized grafts. However, 1 year after the operation, the graft diameter was comparable and equally reduced after the first anastomosis in both groups. Skeletonization with an ultrasonic scalpel increases the luminal diameter of the radial artery graft at early postoperative period, which, however, reduces possibly as adaptation to graft flow 1 year after the operation.The radial artery is increasingly used as a second arterial conduit for myocardial revascularization. However, the radial artery is susceptible to vasospasm, which is thought to be the principal cause of graftfailure. The radial artery is harvested as a skeletonized or a non-skeletonized graft, but the effect of different harvesting technique remains unknown. In this study, we compared the early- and mid-term angiographic findings to elucidate its influence on the graft luminal diameter. We harvested 39 radial arteries either as a skeletonized ( Arterial grafts in coronary artery bypass grafting (CABG) are increasingly utilized since the superior long-term patency of internal thoracic artery (ITA) grafts was reported . CurrentAlthough recent reports have shown better outcomes of RA grafts in comparison to SVG, most of those demonstrated its inferiority to ITA . ProlongThe aims of this study are to compare the early- and mid-term angiographic findings of the RA graft harvested either as a skeletonized or a non-skeletonized graft and examine the influence of the harvesting technique on the luminal diameter of the RA graft.Between July 2008 and September 2009, 39 patients underwent CABG using a straight composite graft constructed with a right internal thoracic artery (RITA) and RA. They underwent postoperative angiography within 1 month after the operation. In 18 patients RA was harvested as a skeletonized graft (Group S) and in the other 21 patients RA was harvested as a non-skeletonized graft (Group N) depending on operators’ preference. All patients were given informed consent about the study. Data were retrospectively collected, from medical records and image servers. Therefore, approval from our hospitals’ institutional review boards was unnecessary.All patients had preoperative examination of the RA by a duplex scan and the preoperative diameter of the RA (preRAD) was determined at 10 mm distal from its proximal end. The RA was harvested from the arm of the non-dominant hand. Skin incision was made from the wrist to the mid-antecubital fossa. RAs were harvested either as a skeletonized or a non-skeletonized graft. In both methods, an ultrasonic scalpel  was used for harvesting. The branches of the RA were cut with protein coagulation by ultrasonic scalpel, and surgical clips were applied only to major branches when necessary in both procedures. For skeletonization of the RA, we incised the fascia covering the RA entirely with scissors at first, and the space between the RA and satellite veins was dissected using an ultrasonic scalpel. A non-skeletonized RA was dissected together with a satellite vein and surrounding connective tissue and finally the back of covering fascia was incised longitudinally with scissors. In both methods, the RA was wrapped with gauze soaked with phosphodiesterase inhibitor solution  until it was removed. After harvesting, the RA graft was flushed and stored in the same olprinone hydrochloride solution.After harvesting the RA, CABG was performed with or without cardiopulmonary bypass. In all the patients, the left internal thoracic artery (LITA) was anastomosed to the left anterior descending artery. RA was anastomosed with a skeletonized RITA as a composite straight graft. We anastomosed both grafts with minimal cut-back at the end of RITA graft. We did not observe any stenosis or kinking at anastomosed site in all patients. RA was sequentially anastomosed to more than 1 native coronary artery segment  with diamond fashion. Severity of angiographic stenosis of the first and the second target vessel was not significantly different between the groups. We started continuous infusion of Nicorandil during graft anastomosis and continued in ICU, which was switched to oral administration of Isosorbide Mononitrate, once oral intake became possible. Calcium antagonist was not routinely used.Postoperative angiography was performed within 1 month after the operation. The postoperative diameter of the RA was measured at 10 mm distal from the anastomotic site with the RITA (postRAD), at 10 mm proximal to the first (RAD1) and the second (RAD2) anastomotic sites, respectively. The postoperative diameter of the RITA was measured at 10 mm proximal from the anastomotic site with the RA (RITAD). These angiographic vessel diameters were calculated using the size of a catheter in the same field as a reference. Flow competition was defined as a situation in which the target branch was slightly opacified or was not opacified from the ITA injection, and the bypass graft was opacified by retrograde flow from the native coronary injection.Twenty-seven patients (15 patients in Group S and 12 patients in Group N) underwent follow-up 320-slice CT angiography  1 year after the operation. Retrospective ECG-gated conventional enhanced 320-slice CT was performed with a slice thickness of 0.5 mm. Contrast material was administered into the right antecubital vein. All patients received 10 mg of propranolol prior to scanning, except for those who had severe heart failure, bronchial asthma, hypotension, or bradycardia due to conditions such as atrioventricular block or sick sinus syndrome. Patients were excluded from this procedure if they did not provide consent or had renal failure. Evaluation of the RA on CT angiogram was performed using a workstation  .t test as appropriate. Differences were considered significant with P < 0.05. All statistical analyses were performed using the JMP, version 11.0.0 .Data are expressed as mean ± SD. Risk factors, patency rates, and other parametric data were examined with contingency tables, Fisher’s exact test, the Chi-squared test, or Student’s There was no statistically significant difference in the two study groups with respect to variables that are known to influence operative outcomes Tables , 2. TherOn angiography at early postoperative period, two grafts were occluded in Group S and all the grafts were patent in Group N. Two grafts showed string sign in Group N, but there was no string sign in Group S. Flow competition was seen less often in Group S although that did not reach statistical significance. Perfect patency rate (patency rate without stenosis >50 % or flow competition) was not significantly different between the groups Table .Table 3EThe mean diameter of the RA on angiogram at early stage after the operation (postRAD) was not significantly different between the two groups. However, the relative size to the preoperative diameter of the RA (postRAD/preRAD) was significantly larger in Group S Table , Fig. 1.On 320-slice CT angiography 1 year after the operation, frequency of graft occlusion or string sign was not significantly different between the two groups Table . The sigThe significant findings of this study are as follows. (1) In the early stage after the operation, skeletonized RA grafts maintained larger diameter in compared to non-skeletonized grafts. Unlike non-skeletonized grafts, skeletonized grafts did not show remodeling of the graft diameter. (2) In the mid-term follow-up CT angiogram, the skeletonized RA graft changed its diameter. There was no difference in this remodeling capacity between the skeletonized and the non-skeletonized graft.Our first finding is consistent with several past reports. Yamaguchi et al.  reportedIt has been well known that arterial grafts change its diameter depending on flow requirement . The preThere are several points that could have influenced our results. First, RA straight composite graft with ITA can behave differently from that used as aortocoronary bypass. Composite grafting allows an aorta no touch technique when combined with off pump CABG, which has major advantage of decreasing perioperative stroke. On the contrary, decreased drive pressure in the composite graft compared to direct aorta anastomosis could compromise the flow through the graft , 22. TheSecond, multiple sequential grafting may also affect the fate of arterial grafts. There are conflicting reports on graft patency between sequential and single RA grafting. Nakajima et al.  reportedThird, it has been well known that arterial grafts change its diameter depending on severity of target vessel stenosis . In our This study has several limitations. First, the number of the patients in each cohort was small and selection of each harvesting methods was not randomized. Although the patient backgrounds were not significantly different between the groups, there was possible selection bias. Second, we could not assess several patients 1 year after the operation. Therefore, we emphasize the need for a large, randomized study.In conclusion, at an early stage after the operation, the RA graft skeletonized with an ultrasonic scalpel maintains larger diameter irrespective of graft flow. It may contribute to avoid graft failure or flow competition in early postoperative period, when the grafts are especially susceptible to spastic stimuli. However, 1 year after the operation, luminal diameter of the skeletonized radial artery graft reduces possibly as physiological adaptation to graft flow. Further studies will be needed to confirm whether those characteristics of skeletonized radial artery graft result in any beneficial short- and long-term graft function.


The purpose of this study was to evaluate the results of hepatectomy with inferior vena cava or hepatic vein resection, followed by vessel reconstruction with an artificial vascular graft.From 2000 to 2011, 1,434 patients underwent several types of hepatectomy at our institution. Of these, we reviewed the cases of eight patients (0.56%) who underwent hepatectomy with inferior vena cava or hepatic vein resection and subsequent reconstruction using an expanded polytetrafluoroethylene (PTFE) graft.We resected the inferior vena cava in six patients and the hepatic vein in two patients. All eight patients underwent subsequent reconstruction using an expanded PTFE graft. The median operative time was 443 minutes and the median blood loss was 2,017 mL. The median postoperative hospital stay period was 18.5 days and the in-hospital mortality rate was 0%. Complications occurred in four patients: two patients experienced bile leakage, one experienced a wound infection, and one experienced pleural effusion. The two patients who experienced bile leakage had undergone reoperation on postoperative day 1. No complication with the artificial vascular graft occurred in these eight cases. Histological invasion to the replaced inferior vena cava or hepatic vein was confirmed in four cases. All artificial vascular grafts remained patent during the observation period.Hepatectomy combined with inferior vena cava or hepatic vein resection, followed by reconstruction with an expanded PTFE graft can be performed safely in selected patients. Liver resection is often the only valid treatment for patients with hepatic tumors such as hepatocellular carcinomas, cholangiocarcinomas and metastatic hepatic carcinomas. Whether hepatectomy can be performed often influences the prognosis of these patients. If the liver tumor is otherwise unresectable, liver surgery requiring venous resection and reconstruction may be the only chance for a cure. Liver tumors invading the inferior vena cava (IVC) or hepatic vein (HV) roots were previously a contraindication for hepatectomy because of the high surgical risk, but along with the progress in liver transplantation, complex hepatectomies, such as those with vein reconstruction, have also become relatively safe. When a In this study, we reviewed the cases of eight patients who underwent hepatectomy with IVC or HV resection and subsequent reconstruction using an expanded polytetrafluoroethylene (ePTFE) graft between 2000 and 2011. The results validate the use of the artificial vascular graft in liver surgery.From 2000 to 2011, 1,434 patients underwent several types of hepatectomy at the Department of Gastroenterological Surgery I, Hokkaido University Hospital. Eight of these patients (0.56%) underwent hepatectomy with IVC or HV resection and subsequent reconstruction using an ePTFE graft . Six of these patients needed IVC resection and two needed HV resection. The clinical and surgical features of the patients are listed in Table Preoperative management was done according to our previous report. All pat2O to prevent venous hemorrhage. Liver resection in this study included two right trisectionectomies, two left hepatectomies, one right hepatectomy, and three partial resections.The surgical method used for the liver resections has been previously described. Transecn = 1). When the defect of the IVC wall was greater than one-half of the entire circumference, the IVC was reconstructed with a 20 mm ringed ePTFE tube graft  or from the middle hepatic vein (MHV) to the IVC (patient 6). In reconstructing the IVC with an ePTFE graft, the IVC was clamped as follows: the IVC was clamped at both the suprarenal and suprahepatic regions, the liver and invaded IVC was removed, and the IVC was reconstructed with an ePTFE graft. After reconstruction, the IVC clamp was first released at the suprarenal region and later at the suprahepatic region for the air flush. In reconstructing the HV with an ePTFE graft, the IVC was clamped as follows: the IVC was clamped at both the suprarenal and suprahepatic regions and inflow occlusion was applied, and the HV was reconstructed with an ePTFE graft. After HV was reconstructed and inflow occlusion was released, the IVC clamp was first released at the suprarenal region and later at the suprahepatic region for the air flush. At our institution, venovenous bypass is used in case of a decrease in systolic blood pressure of less than 60 mmHg by test clamping the IVC even after volume loading. A venovenous bypass was not used for any the patients in this study because no cases had a decrease in blood pressure when clamping the IVC, although we were prepared to use the bypass.No anticoagulant therapy was used for patients undergoing reconstruction of the IVC with an ePTFE graft. Anticoagulant therapy with warfarin was used to maintain an international normalized ratio of 1.5 to 2.0 for patients undergoing reconstruction of the HV with an ePTFE graft. Ultrasound sonography was performed daily for one week after surgery to check the graft patency. CT was performed on postoperative day 7 to evaluate the graft patency, and follow-up studies after discharge were conducted one month after the operation and at three-month intervals thereafter.The details of the surgical results are listed in Table We examined the cases of eight patients who underwent liver surgery combined with IVC or HV resection, followed by vessel reconstruction with artificial vascular grafts, and explored the validity of the use of ePTFE grafts in liver surgery. There were no surgical complications accompanying the use of artificial vascular grafts and all grafts in patients were patent. Therefore, liver resection combined with vessel reconstruction of the IVC or HV with subsequent reconstruction using ePTFE grafts can be performed safely in selected patients. Furthermore, our series contained two cases of over five-year survival Table , althougIn liver surgery, there are a few cases that can only be cured by performing resection and reconstruction of the IVC or HV. Primary closure is impossible, and patch closure is often difficult during IVC reconstructions when the tumor invasion range is large; thus, reconstruction with an artificial vascular graft is often required. Experieet al.[et al.[et al.[In the cases presented here, complications occurred in four patients: two patients experienced bile leakage, one experienced a wound infection, and one experienced pleural effusion. However, there were no complications involving the ePTFE grafts. Graft infection is the most serious complication related to the use of ePTFE grafts. In our series, biliary reconstruction by Roux-en-Y hepaticojejunostomy was performed in one case and reoperations were performed for bile leakage on postoperative day 1 in two cases, but graft infection did not occurred in any of the patients. Arii et al. reported[et al. reported[et al. reported[et al.. As seen[et al.,15. Howe[et al.. One gra[et al.. Therefoet al.[Another important issue with ePTFE grafts is graft patency. In our series, all the cases showed graft patency throughout the observation periods. In liver surgery using an ePTFE graft for IVC reconstruction, the ePTFE graft has been reported to show very high patency rates,11,12,18et al. reportedet al.. Therefoet al.. Use of et al.. In thiset al.. Anotheret al.. Luminalet al.. Althouget al.[ex vivo resection techniques if there was complex involvement of the IVC, hepatic veins, and/or portal structures. Ex vivo resection is rarely needed because most tumors can be resected with less technically demanding techniques; however, ex vivo resection may be effective for patients in whom obtaining tumor-free margins during an in situ aggressive surgical procedure is not possible.The important feature of liver resection using an artificial vascular graft is to secure tumor-free margins for patients in whom tumor-free margins cannot be obtained with standard liver resection. Recently, Hemming et al. reportedHepatocellular carcinomas often expand with a capsule and form a tumor thrombus into intrahepatic vessels rather than directly invading the vessels. By contrast, adenocarcinomas, such as cholangiocarcinomas and metastatic tumors from colon cancer, tend to invade surrounding organs directly. BecauseOur results indicate liver resection combined with IVC or HV vessel reconstruction using an ePTFE graft can be performed relatively safely in selected patients. Furthermore, precise preoperative assessment and surgical planning are mandatory to safely perform liver surgeries requiring venous reconstruction.3D-CT: three-dimensional computed tomography; CT: computed tomography; ePTFE: expanded polytetrafluoroethylene; HV: hepatic vein; ICGR15: indocyanine green retention rate at 15 min; IVC: inferior vena cava; LHV: left hepatic vein; MHV: middle hepatic vein.The authors declare that they have no competing interests.TO and TK designed the research. TO, TK, HY, TK, KW, YT, HK, and AT contributed to acquisition of data. TO, TK, and AT analyzed and interpreted data. All authors read and approved the final manuscript.