Virgin olive oil (VOO) is credited as being one of the many healthful components associated with the Mediterranean diet. Mediterranean populations experience reduced incidence of chronic inflammatory disease states and VOO is readily consumed as part of an everyday Mediterranean dietary pattern. VOO is rich in phenolic compounds and the health promoting benefits of these phenolics are now established. Recent studies have highlighted the biological properties of VOO phenolic compounds elucidating their anti-inflammatory activities. This paper will review current knowledge on the anti-inflammatory and nutrigenomic, chemoprotective and anti-atherosclerotic activities of VOO phenolics. In addition the concentration, metabolism and bioavailability of specific phenolic compounds will be discussed. The evidence presented in the review concludes that oleurepein, hydroxytyrosol and oleocanthal have potent pharmacological activities in vitro and in vivo; however, intervention studies with biologically relevant concentrations of these phenolic compounds are required. The health promoting attributes associated with following a traditional Mediterranean diet have been reviewed extensively. The first suggestion of healthful effects arose from the Seven Countries Study conducted in the 1970’s . As a reThe principle source of dietary fat in the Mediterranean diet is VOO ,19 and VIt is now known that VOO contains approximately 36 phenolic compounds and these compounds play a key role in the health promoting qualities of the oil ,24,25,26Over 30 VOO phenolic compounds have been identified and considerable variation has been noted in the concentration of such phenolic compounds (0.02 to 600 mg/kg) . VOO pheA number of factors have been shown to affect the concentration of phenolic compounds in VOO and these include olive fruit cultivar and variety, region of growth, agricultural cultivation techniques, maturity at harvest, processing, and storage. Also, olive tree age has been shown to influence phenolic content . The phenolic concentration and composition of VOO together with the degree to which these components are absorbed and metabolized are essential in determining the health effects associated with such compounds. Bioavailability of VOO phenolic compounds is the key in achieving an effect in specific tissues or organs .The majority of research regarding the bioavailability of these compounds has focused on the two most abundant VOO phenolics: hydroxytyrosol and tyrosol, amongst a few others . ResearcWith regards to the metabolism of VOO phenolics, the presence of metabolites from the majority of olive oil phenolic compounds  in human urine has been noted . This imVOO intake for Mediterranean populations is reported to be 30–50 g/day . If we aIt is known that the pathophysiology of common disease states such as cancer, CVD, arthritis and neurodegenerative disease takes root in chronic inflammation . PhenoliThere are now several studies that have examined the anti-inflammatory and cardio-protective effects of olive oil phenolic compounds in humans. These studies suggest that VOO with high phenolic concentration is effective in modulating inflammatory mediators derived from arachidonic acid, such as thromboxane B2 and 6-keto-PG F1a, as well as other inflammatory markers, such as high-sensitivity C-reactive protein (CRP) and interleukin 6 (IL-6) ,43,44. MIn regard to the specific actions of the phenolic oleuropein, early evidence demonstrates this phenolic inhibits tumour necrosis factor alpha (TNFa) induced matrix metalloproteinase 9 (MMP-9) in a monocyte cell line. This has implications for health as monocytes, and the molecules they secrete play a significant role in inflammatory disease development . FurtherFurthermore, the phenolic compound oleocanthal has been found to share the same mechanistic anti-inflammatory pathway as the non-steroidal anti-inflammatory drug, ibuprofen. In vitro, oleocanthal has been shown to inhibit both cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) inflammatory enzymes in a dose-dependent manner, and is more effective than ibuprofen in inhibiting these enzymes at equimolar concentrations . The antFurther evidence suggests that oleocanthal may be a potent pharmacological agent in the treatment of neurogenerative disease, as this compound exhibits neuroprotective properties, in addition to attenuating markers of inflammation implicated in Alzheimer’s disease ,51,52,53Hydroxytyrosol has been reported to possess significant anti-inflammatory actions in an animal model of inflammation and to attenuate TNFa and interleukin 1β (IL-1b) expression, which are pro-inflammatory cytokines often observed in inflammatory disease  FurthermDue to the anti-inflammatory efficacy of VOO phenolics, much research has focused on the pharmacological properties of these compounds, in particular the anti-carcinogenic actions. There is much research suggesting that oleuropein and its metabolite, hydroxytyrosol, exert anti-cancer properties. An early study has shown that high doses of oleuropein at a concentration of 200 μg/mL decreased cell viability and inhibited cell proliferation in human breast adenocarcinoma (MCF-7) breast cancer cells . FurtherChimento and colleagues  report tA recent study showed that oleocanthal inhibits the mammalian target of rapamycin (mTOR) . mTOR isEarly research has shown that VOO phenolics improve endothelial dysfunction and reduce oxidative stress plasma parameters ,75, whicFurthermore, a recent randomized control trial reported that VOO phenolic intake decreased low density lipoprotein (LDL) concentrations and LDL atherogenicity in vivo . This stVOO phenolics have been shown to influence the expression of genes related to obesity. Warnke and colleagues observed that hydroxytyrosol at a concentration of 25 μM was able to modify genes related to adipocyte maturation and differentiation and to have an inhibitory effect on lipid formation . HyperliIn an animal model of high fat diet-induced obesity, hyperglycemia, hyperlipidemia, and insulin resistance, it was shown that hydroxytyrosol decreased high fat diet-induced lipid deposits. The mechanism of action was the inhibition of the SREBP-1c/FAS pathway in liver and skeletal muscle tissues, an increase in antioxidant enzyme activities, and the normalization of the expression of mitochondrial complex subunits and mitochondrial fission marker Drp1 . FurtherThese data indicate that VOO phenolics may have a protective role against excessive fat accumulation associated with systemic oxidative stress as well as exert beneficial effects against the development of metabolic syndrome. However, clinical studies are needed to support the evidence from in vitro and animal models. To conclude, the beneficial effects of VOO phenolics on health have been researched extensively, and recent research supports earlier evidence that these components exert beneficial effects on physiological processes related to health and disease. A number of studies both in vivo and in vitro demonstrate that VOO phenolic compounds beneficially alter inflammation and have beneficial effects on markers of cancer, atherosclerosis and also genes related to obesity and metabolic syndrome. The modes of action through which olive oil phenolic compounds beneficially influence health parameters detailed in this review may explain the lower incidence of chronic inflammatory diseases amongst populations residing in the Mediterranean region, who consume large volumes of VOO in their daily diets. The evidence presented in the review concludes that oleuropein, hydroxytyrosol and oleocanthal possess potent pharmacological activities in vitro and in vivo. This expanding collection of evidence would further benefit from human intervention studies with biologically relevant concentrations of these phenolic compounds.


The chromatin landscape has acquired deep attention from several fields ranging from cell biology to neurological and psychiatric diseases. The role that DNA modifications have on gene expression regulation has become apparent in several physiological processes, and numerous efforts have been performed to establish a relationship between DNA modifications and physiological conditions, such as cognitive performance and aging. DNA modifications are incorporated by specific sets of enzymes—the writers—and the modified DNA-interacting partners—the readers—are ultimately responsible for maintaining a functional epigenetic landscape. Therefore, understanding how these epigenetic mediators—writers and readers—are modulated in physiological aging will contribute to unraveling how aging-associated neuronal disturbances arise and contribute to the cognitive decline associated with this period of life. In this review, we focused on DNA modifications, writers and readers, highlighting that despite some methodological disparities, the evidence suggests a critical role for epigenetic mediators in the aging-associated neuronal dysfunction. Changes in gene expression allow the dynamic nature of the neuronal function. The role of gene expression regulation in directing neuronal states has been highlighted in synaptic plasticity and learning and memory processes . HoweverOne of the mechanisms through which gene expression is controlled in a long-lasting manner is by the remodeling of the chromatin landscape induced by epigenetic changes, which are chemical modifications in histone proteins and DNA bases. Modifications of the chromatin landscape are functionally relevant, as it settles the accessibility of protein complexes to regulatory regions of promoters and thus direct gene expression. The epigenetic modifications are signatures through which environmental factors chemically modify the genome to leave a record and form, a many times permanent, genetic-molecular memory. In this regard, epigenetic modifications have emerged as a relevant mechanism underlying dynamic transcriptional regulation contributing to neuronal function , and sevBesides those changes in histone and DNA methylation/hydroxymethylation, aging-associated changes in epigenetic mediators—writers and readers—could be involved in the age-related changes in gene expression that underlie one of the most singular characteristics associated to this period of life, the cognitive decline. Moreover, the processes leading to epigenetic mark erasure could also contribute to aging-associated disturbances and should be considered. Therefore, a clear understanding of the age-associated expression dynamics of epigenetic writers and readers will contribute to unraveling the aging-associated disturbances of neuronal function. In this review, we focused on DNA methylation and DNA hydroxymethylation writers and readers to highlight our current knowledge of how physiological aging modifies the expression of epigenetic mediators and the relevance of these changes for the maintenance of cognitive abilities.maintenance DNMTs and de novo DNMTs. Both groups share the use of S-adenosyl-L-methionine (SAM) as a methyl donor, and both groups of DNMTs are expressed in the brain [maintenance DNMT and is proposed to maintain DNA methylation after DNA replication [de novo DNMTs and incorporate a previously nonexistent 5meC to the genome. The DNMT3a locus gives rise to 2 protein isoforms named DNMT3a1 and a DNMT3a2. The DNMT3a2 lacks 219 aminoterminal residues in comparison to DNMT3a1, and it has been shown to be sensitive to an increase in neuronal activity [The covalent binding of a methyl group to carbon 5 of a cytosine (5meC) is an epigenetic mark that is subjected to dynamic modifications. 5meC occurs at a higher frequency in cytosines of CpG dinucleotides; however, 5meC in non-CpG dinucleotides has acquired an increased relevance . 5meC ishe brain , 10. DNMlication . On the activity . In addiactivity . Non-CpGactivity , 14. Intactivity –17.α-ketoglutarate and Fe (II)-dependent reaction, generating an interesting link to cellular metabolism [5meC can undergo an active series of chemical modifications that lead to cytosine demethylation. This pathway involves the oxidation of methylcytosine to hydroxymethylcytosine (5hmeC), 5-formylcytosine (fC), and 5-carboxylcytosine (caC) followed by excision of fC or caC mediated by thymine DNA glycosylase coupled with base excision repair . The firtabolism . TET enztabolism –22.5meC regulates gene expression either directly, by inhibiting the association of transcription factors to its binding site, or indirectly via a family of proteins containing a methyl-binding domain (MBD) . The MBDThe mechanism through which Mecp2 activates the expression of its target genes is still a matter of study. It has been described that Mecp2 interacts with the cAMP response element-binding protein (CREB) , a widelIt has also been shown that Mecp2 binds 5hmeC in the central nervous system ; therefoInterestingly, neuronal activity modulates Mecp2 phosphorylation, decreasing phosphoserine 80  and incrAs exemplified by Mecp2 phosphorylation, there is a vast range of posttranslational modifications regulating the function of Mecp2 . UnderstThe information regarding Mecp2 contribution to cognitive function comes mainly from studies using mouse models of RTT. For instance, a mouse model containing a truncated form of Mecp2 shows impaired synaptic plasticity and learning and memory . SimilarThe cognitive decline associated with aging is linked to a change in gene expression that drives several neuronal functions , 46. As Although few efforts have been made to elucidate the role of epigenetic mediators in aging, the results obtained have been highly interesting and call for further attention. Based on the general consensus that global DNA methylation decreases in the aged brain , OliveirA reduction in hippocampal TET1 and TET2 expression was observed in 18-month-old mice . ConcordAs was mentioned above, MECP2 binds to either 5mC or 5hmC and mutation in its coding gene is the main cause of RTT. Since RTT symptoms arise early in development, most of MECP2 literature overlooks the role for this protein in aging. However, tissue microarray analyses showed that MECP2 expression increases along aging in humans , 60 and Some environmental conditions attenuate or delay characteristics associated to physiological aging. Among those conditions, physical exercise (PE), caloric restriction (CR), and environmental enrichment (EE) have been shown to modulate the epigenome and contribute to improve cognitive function –68. For in vitro, counteracting the diminished DNMT1 expression reported for aging [in vitro and it has been shown that EE reduced Mecp2 mRNA expression in 8-week-old mice [Although intrinsic age-associated disruption of neuronal circuits occurs during aging , the cogor aging . Interesold mice , 80, whiold mice –61. Suppold mice . TherefoThe aging-induced reduction in neuronal stimulation adds a layer of complexity to the interpretation of aging-related changes in gene expression. Notwithstanding, the relevance of epigenetic mediators in aging has been strikingly highlighted by some of the works reviewed, raising an urge to fulfill the voids in our understanding.As it was discussed above, physiological aging is accompanied by a cognitive performance decline associated with changes in the expression of epigenetic writers and readers that modify chromatin landscape and thus impact on gene expression. However, more evidence is required to fulfill the gaps among all these processes and take our knowledge beyond correlations between these aging-associated events. To achieve that, it is needed to unveil the mechanism underlying the changes in the expression of DNA readers and writers and the physiological factors associated with the signaling that leads to these modifications. Since the transcriptional changes that accompany aging seem to be gene-specific and do not affect the entire genome, it is critical to identify the mechanism through which DNA writers select their target regions to write on it. Then, with that knowledge, we could understand how through environmental factors these changes can be prevented or mitigated to maintain a better cognitive performance during aging, which is critical nowadays in which the life expectancy has raised.


These processes of dissolution and entrapment can result in the formation of rock coatings that contain elevated levels of heavy metals. Between 1927 and 1976, the Horne smelter processed sulfide ore derived from the Rouyn-Noranda region and became one of the largest emitters of particulates and sulfur dioxide in North America, promoting the formation of coatings on nearby rock surfaces. The reflectance spectra of these coatings are relatively flat, with typical reflectance values ranging between ~5% at visible wavelengths and ~16% in the shortwave infrared. Absorption troughs in coating spectra are consistent with the presence of materials including opaline silica, olivine, pyroxene, hydrous phyllosilicates, and sulfates. Classification of Landsat 8 Operational Land Imager data indicates that rock coatings near Rouyn-Noranda comprise a total surface area of ~1.5 km2, nearly all of which is located within ~6 km of the Horne smelter. Remote sensing techniques can used to delineate the geographic extents of coatings near smelters, highlighting areas previously subjected to severe environmental degradation.Emissions of SO Also, emissions of sulfur dioxide from smelters can promote formation of acid rain, which along with other types of emissions can impact local vegetation health and reduce vegetation cover6. Under extreme conditions, acid rain can dissolve siliceous minerals on exposed rock surfaces and thereby promote the formation of thin silica gel layers within which detrital and smelter particulates can become trapped9. Over time, these processes of dissolution and entrapment can result in the formation of brownish-black rock coatings that contain components such as oxides, sulfates, high-temperature silicates, carbon-rich particulates, and rock and soil particulates; and that are characterized by high levels of heavy metals10.The smelting of sulfide ores can produce atmospheric emissions rich in particulates and heavy metals12. Related techniques may also prove useful in the monitoring of environmental recovery where smelter emissions have been reduced. In prior work, the supervised classification of Landsat Enhanced Thematic Mapper Plus (ETM+) and Hyperion satellite images was successfully used to determine the spatial distribution of rock coatings near three smelting sites in the Sudbury region of Ontario, Canada, highlighting areas previously exposed to relatively high levels of acid rain and smelter-derived particulates12.The use of remote sensing techniques in the detection and mapping of rock coatings near smelters can help to delineate the geographic extents of areas subjected to past environmental degradation, and has the potential to be applied toward the monitoring of ongoing degradation at sites where emissions continue to promote the development of coatings14, negatively affecting local vegetation vigor and extent16 and contaminating surface materials with heavy metals19. Over time, coatings formed on exposed rock surfaces proximal to the Horne smelter20.This study extended the scope of earlier work at Sudbury, Ontario, to the Rouyn-Noranda region of Quebec Fig. , in ordeIn this study, the reflectance spectra of rock coatings from the Rouyn-Noranda region were measured and described, and were compared with those previously collected for the Sudbury region. Also, a reflectance database derived from Landsat 8 Operational Land Imager (OLI) data for this region was classified in order to determine the spatial distribution of rock coatings produced by the Horne smelter, and to further evaluate the utility of remote sensing techniques in the study of environmental degradation caused by smelters.22. Further to the south are predominantly sedimentary units of the Timiskaming and Cadillac groups , the Cobalt Group , and the Pontiac Group (a northern unit of the Pontiac Subprovince)24  and the presence of turbidite and argillite interbeds30. Volcanic units of the Blake River Group were intruded by several generations of plutons, dikes, and sills26, with the most prominent intrusions including the Flavrian and Powell tonalites and the Lac Dufault granodiorite31  deposits37. Topographic relief here is less than 150 m, and bedrock exposure is discontinuous. Glacial lakes Ojibway and Agassiz-Ojibway extended across much of western Quebec in the Early Holocene, and glaciofluvial, glaciolacustrine, and glacial till deposits are variously distributed across parts of the study area39. The study area falls within the balsam fir and white birch domain of the boreal vegetative zone, with common tree types including white spruce, black spruce, balsam fir, aspen, birch, and jack pine40. Lichen cover can be extensive on local bedrock41.The study area is located in the boreal ecoclimatic region of Canada14. Metals released into the atmosphere during and following this period have most notably included lead, zinc, nickel, arsenic, copper, and cadmium47. Annual emission of particulate matter here peaked in 1965 at more than 1.5 million metric tonnes13. Pollution controls were implemented at the Horne smelter beginning in the 1970s, resulting in reductions in total emissions14. The Horne smelter ceased the processing of local sulfide ores in 1976, and over the following decades became a processor of electronic scrap containing copper and precious metals.The Horne smelter Figs  and 3A p48. In areas proximal to the Horne smelter, such emissions have previously stunted the growth of trees16 and affected the abundances of some varieties of vegetation . Smelter emissions may have also increased the acidity of some lakes in the region49. Soils, peat hummocks, aquatic organisms, and seasonal snow in the Rouyn-Noranda region have contained elevated levels of heavy metals both during and following the period of peak annual emissions, with metal concentrations generally decreasing with increasing distance from smelter stacks51.As with emissions of particulate matter, annual emissions of sulfur dioxide peaked in 1965 at about 710,000 metric tonnes, and have steadily declined since that time20. Over time, these processes promoted development of prominent rock coatings with colors that range between black and reddish-brown  data Fig. . Reflect12. In that work, the maximum likelihood algorithm, with null class implemented, outperformed a feedforward backpropagation neural network classifier and the Spectral Angle Mapper classifier by consistently producing useful and representative coating maps characterized by low proportions of false positives. Using the Sudbury results as a basis, the present study focused on classification of the Landsat 8 reflectance database using the maximum likelihood classifier.Prior research demonstrated the effectiveness of the maximum likelihood algorithm in the remote-sensing-based identification of coated rock surfaces in the Sudbury region of Ontario, Canada63. For the utilized version of the maximum likelihood algorithm, the a priori probability of the presence of particular surface classes64 is assumed to be equal for all classes, and individual pixel labels are assigned to the classes that have the highest associated probabilities. The use of a null class ensures that pixels are only labeled if peak probabilities exceed a threshold, which in the utilized algorithm is defined by a hyperellipsoid with a surface located three standard deviations from the mean vector of any given class. This can improve classification outcomes for e.g. the rock coating class by reducing false positive detections in areas where materials with somewhat similar reflectance characteristics are present 12. Though rock coatings were of prime interest in this study, the utilized algorithm requires the specification of a representative and complete list of general surface cover classes. Classifications were produced for the Rouyn-Noranda study area using the following set of classes: coated rock, uncoated rock, uncoated urban materials, uncoated clearings and open pits, predominantly coniferous vegetated sites, predominately deciduous or grassy vegetated sites, deeper water, and shallower or turbid water.Among the most widely used routines for supervised classification, the maximum likelihood algorithm parameterizes the ranges of satellite image values that characterize the training sites of particular classes using Gaussian probability distributions derived from associated mean vectors and matrices of covariance20. Quantitative evaluations of surface cover data were also performed, based on comparison of classification outcomes with test pixels known to be dominated by particular surface cover classes. The numbers of test pixels used in this study were as follows: coated rock (180 test pixels), uncoated rock (288 test pixels), uncoated urban materials (519 test pixels), uncoated clearings and open pits (434 test pixels), predominantly coniferous vegetated sites (349 test pixels), predominantly deciduous vegetated sites (391 test pixels), deeper water (168 test pixels) and shallower or turbid water (255 test pixels).Classification of the Landsat 8 reflectance database allowed for the generation of a map of exposed rock coatings for the Rouyn-Noranda study area. Predicted sites of extensive rock coatings were evaluated in terms of their qualitative consistency with field-based knowledge of the study area gained in this study and in a previous studyResults generated in this study, including those related to both the spectral characterization of Rouyn-Noranda rock coatings and the generation of a map of prominent rock coatings in the study area, are given below.Reflectance spectra of selected coated and uncoated rock surfaces from the Rouyn-Noranda study area are given in Fig. 66, which are high-temperature minerals expected to comprise some smelter-derived spheroidal clasts embedded within the silica-rich matrix of coatings. Absorption features near 1400 and 1900 nm suggest the presence of water67, which is consistent with the expected presence of constituents such as opaline silica as well as hydrous phyllosilicates and sulfates. Features near 2200 and 2350 nm are consistent with the presence of OH-bearing minerals including clays, iron hydroxides, and iron sulfates69. Absorption near 2350 nm is also consistent with the presence of carbonates70, but minerals of this class are not expected in significant amounts due to the relatively acidic conditions that were present during coating formation. Absorption features such as those near 900, 2200, 2260, 2325, and 2350 nm are consistent with the presence of minerals including sulfates72, which are confirmed constituents of some coating surfaces and can also be present within coatings and along the interfaces between coatings and underlying substrates  were correctly identified as such, 19 of 180 coated bedrock test pixels (10.5%) were not classified and were instead assigned to the null class, and only 20 of 180 coated bedrock test pixels (11.1%) were misclassified as uncoated (with 12 of 180 misclassified as uncoated bedrock and 8 of 180 misclassified as uncoated urban) , this gives a total predicted surface area of 1,158,300 m2, or ~1.2 km2. A classification success rate of 78% for the coated bedrock class (Section 7.2) implies a current total surface area of rock coatings of ~1.5 km2. Though notable, the spatial extent of prominent dark coatings in the Rouyn-Noranda study area is only roughly 1% of that which characterizes the Sudbury region11, where emissions from historical roast yards and three major smelting centers prior to the mid-1970s produced a total surface area of exposed rock coatings that is estimated for the year 2000 to have been as great as ~155 km2.A total of 1287 pixel locations are predicted to have been associated with well-exposed fully or partly coated bedrock in the Rouyn-Noranda study area in 2017. With pixel areas of 30 × 30 m  can be subtle and are less likely to be consistently detectable using image databases with poorer spectral characteristics.This study utilized Landsat 8 data characterized by a relatively coarse pixel size of 30 × 30 m. Improved outcomes in the discrimination of rock coatings may be possible, especially where coatings are notably discontinuous and irregularly distributed, through the use of image databases with higher spatial resolutions. In such areas, smaller pixel sizes may increase the abundance of relatively pure pixels , possibly providing a clearer basis for discrimination of the coated class. Improved classification outcomes may also result from the use of hyperspectral rather than multispectral image databases, since differences in the reflectance properties of the coated class and certain uncoated classes (e.g., mine tailings and asphaltClassification results for the Rouyn-Noranda study area confirm that multispectral datasets can be used to detect and map prominent rock coatings near smelters, highlighting the overall spatial distribution of sites previously subjected to severe levels of acid rain and smelter-derived particulates. Remote sensing techniques should be applicable to the study of the spatial distributions of rock coatings at smelting sites beyond those of Sudbury and Rouyn-Noranda, including sites where environmental conditions are sufficiently severe for the development of rock coatings to be ongoing. For example, a time series of maps of rock coatings could potentially be used to better understand the spatial distribution of ongoing environmental degradation in the vicinities of smelters that are actively emitting high levels of particulates, heavy metals, and sulfur dioxide. Remote sensing techniques may similarly prove useful in the monitoring of environmental recovery where smelter emissions have been reduced or eliminated, by facilitating the generation of maps that depict the progressive reduction of exposed coatings as a result of weathering and erosion, revegetation, and the construction of roads and other infrastructure.Prior to the mid-1970s, development of rock coatings was promoted in the Rouyn-Noranda region by emissions of particulates and sulfur dioxide from the Horne smelter. Though typically less than 50 micrometers thick, these coatings are generally very effective at masking the reflectance characteristics of rock substrates.Overall, coatings are similar in composition to those previously investigated in the Sudbury region, and correspondingly have similar spectral characteristics. Rock coatings in the Rouyn-Noranda region have relatively low albedo across the visible, near-infrared, and shortwave infrared, and associated reflectance spectra are characterized by subtle absorption features that are consistent with the known or expected presence of constituents including opaline silica, olivine, pyroxene, hydrous phyllosilicates, and sulfates.Classification of a reflectance database derived from Landsat 8 OLI data successfully produced a map of prominently coated bedrock in the Rouyn-Noranda region. This map does not identify all areas that are less distinctly coated, and thus underestimates the total spatial extent of coated bedrock here. Detection of additional exposures of coated bedrock could not be performed without increasing confusion between coated bedrock and uncoated materials such as mine tailings.2, nearly all of which is located within ~6 km of the Horne smelter.The total surface area of rock coatings near Rouyn-Noranda is estimated to currently be ~1.5 kmImproved outcomes in the discrimination between rock coatings and other classes may be possible through the use of image databases with higher spatial resolutions and/or enhanced spectral characteristics.


During the light response, the Ca2+-free Rcv releases GRK, which in turn phosphorylates visual pigment, ultimately leading to the cessation of the visual transduction cascade. Technological advances over the last decade have contributed significantly to a deeper understanding of Rcv function. These include both biophysical and biochemical approaches that will be discussed in this review article. Furthermore, electrophysiological experiments uncovered additional functions of Rcv, such as regulation of the lifetime of Phosphodiesterase-Transducin complex. Recently, attention has been drawn to different roles in rod and cone photoreceptors.This review article focuses on Rcv binding properties to Ca2+, disc membrane and GRK, and its physiological functions in phototransduction and signal transmission.Recoverin (Rcv) is a low molecular-weight, neuronal calcium sensor (NCS) primarily located in photoreceptor outer segments of the vertebrate retina. Calcium ions (Ca Specialized Ca2+-binding proteins, belonging to the neuronal calcium sensor (NCS) family, detect neuronal Ca2+  spectroscopy  state  state. The Ca2+ induced exposure of the myristoyl group, known as Ca2+ myristoyl switch, allows Rcv binding to the phospholipid membranes only when intracellular Ca2+ concentration ([Ca2+]i) is high  is known as a powerful tool to measure interaction modes of proteins, peptides, and lipids Koch, . The sub2+ and the presence of the myristoyl group  via strain-promoted azide-alkyne cycloaddition. In this way, the membrane translocation of Rcv is visualized in a Ca2+ dependent manner and the membrane with negative spontaneous curvature or higher fluidity shows stronger Rcv binding in the presence of Ca2+.Another useful technique to study the Rcv-membrane interaction is site-specific fluorescent labeling  has been implicated as an internal modulator of Ca2+ sensitivity and therefore may also affect GRK binding  influx across the plasma membrane into the outer segment. This Ca2+ influx is reduced by the closure of the CNG channels during light response, while Ca2+ efflux via Na+/Ca2+ K+ exchanger continues, leading to a decrease in [Ca2+]i in the outer segment. This decline in [Ca2+]i modulates the quenching of the phototransduction cascade, which requires the shutoff of both active intermediates (R* and PDE*) and the resynthesis of cGMP. Under steady background illumination, the [Ca2+]i decline is proportional to the reduction in the circulating current and is crucial for lower photosensitivity and faster response kinetics during this process. What are the targets of Ca2+? The light-induced decrease in [Ca2+]i is postulated to accelerate phosphorylation of R* via Rcv, speed up cGMP synthesis via guanylyl cyclase activating protein (GCAP), and increase cGMP affinity of the CNG channel via calmodulin .Despite the fact that Rcv has been extensively shown to inhibit GRK in a Ca2+ is high, its deletion should result in a shortened lifetime of R* and decrease the gain of signaling pathway. This in turn should lead to lower light sensitivity, without strongly affecting the time course of response decay. However, electrophysiological recordings from Rcv−/− mice showed an accelerated dark-adapted flash response, but only a minor effect on response sensitivity  and the exponential decay constant of the response (τREC) are significantly accelerated in Rcv−/− rods  is slower than R* decay (around 50 ms) and dominates the overall photoresponse recovery in mouse rods .During light adaptation, rod sensitivity is reduced and response recovery is accelerated when background illumination increases, which extends the working range of rods to brighter light levels  may still be involved. Is this mechanism Ca2+ dependent? Background light can still accelerate the response in Rcv−/− rods recorded in low extracellular Ca2+ concentration ([Ca2+]o) which in theory can clamp [Ca2+]i to its maximal light-adapted level, indicating a possible Ca2+-independent process  and the deactivation of PDE*. GTPase-activating proteins (GAPs) accelerate the turn off rate of Transducin. Deletion of GRK in GAPs deficient rods produces little effect on the time course of response, suggesting that the target protein(s) may be involved in the PDE-Transducin pathway  but not saturating light  activity in cones is more than 100 times greater than the range of regulation of rod homolog of GRK (GRK1) activity in rods, suggesting that the phosphorylation mediated by Rcv may contribute more during light adaption in cones  must be at work to regulate the photoresponse, for example via GCAPs , therefore, they presumably work together in a similar way in both cell types. Electroretinography (ERG) on Rcv deficient cones shows not only an accelerated response recovery, but also an around twofold decrease in photosensitivity in darkness, which differs from rods  GRK affinity among Rcvs. Indeed, a recent biochemical work shows different Ca2+ affinity and Ca2+-induced conformational changes among zebrafish Rcv isoforms and bovine Rcv  of Rcv which is independent from GRK in other cellular compartments of photoreceptors.Rcv is expressed in all photoreceptors and also in some subtypes of bipolar cells  and the potential function difference between rod and cone Rcv will need to become a focus of future research.Here, we reviewed the molecular process underlining CaJZ and SN drafted and revised the review article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


L-glutamate is the major excitatory amino acid in the mammalian central nervous system (CNS). This neurotransmitter is essential for higher brain functions such as learning, cognition and memory. A tight regulation of extra-synaptic glutamate levels is needed to prevent a neurotoxic insult. Glutamate removal from the synaptic cleft is carried out by a family of sodium-dependent high-affinity transporters, collectively known as excitatory amino acid transporters. Dysfunction of glutamate transporters is generally involved in acute neuronal injury and neurodegenerative diseases, so characterizing and understanding the mechanisms that lead to the development of these disorders is an important goal in the design of novel treatments for the neurodegenerative diseases. Increasing evidence indicates glutamate transporters are controlled by the circadian system in direct and indirect manners, so in this contribution we focus on the mechanisms of circadian regulation  of glutamate transport in neuronal and glial cells, and their consequence in brain function. Thi the SCN –22, alth the SCN .Glutamate (Glu), the main excitatory neurotransmitter in the mammalian central nervous system (CNS), activates two subtypes of Glu receptors: ionotropic (iGluRs) and metabotropic (mGluRs) –25. The +-dependent high affinity Glu transporters carries out the Glu removal from the synaptic space. The excitatory amino acid transporters (EAATs) comprise five different Glu transporters: Glu/aspartate transporter (GLAST), Glu transporter 1 (GLT1), excitatory amino acid carrier 1 (EAAC1), excitatory amino acid transporter 4 (EAAT4), and excitatory amino acid transporter 5 (EAAT5) or EAAT 1-5 according to rodent and human nomenclature, respectively , as with AD, there is also a decrease in Glu uptake; in PD, Glu transporters have an unusual trafficking between membrane and cytoplasm leading a decrease in Glu transporters at the plasma membrane . This phLikewise, Glu transporters have a critical role in Huntington's disease (HD), in which the expression of these transporters is diminished, the symptoms of HD worsen . In thisIt is well-known that Glu transporters are regulated at different levels, at the transcriptional translational and post translational levels through modifications of transporter protein, as well as by the transporter targeting and trafficking –64. NeveGlast mRNA and protein levels present a diurnal rhythm in 12/12 h light-dark conditions  to ZT18  are a good example of a system that can rapidly respond to external stimuli since it is activated without changes in transcription and/or translation . In thiscis- and trans-elements involved in its transcriptional regulation. Specifically, the circadian regulation of Glu transporters is an emerging theme that promises to be an indispensable tool in the preventing and/or treatment of diseases related to alterations in glutamatergic system. Future research should be directed to study of molecular mechanisms involved in circadian modulation of these transporters.In the last two decades, several research groups have examined the different signaling pathways that modulate glial Glu transporters expression (GLAST and GLT1). Scarce information about EAAC1, EAAT4, and EAAT5 transporters is available. Particularly, EAAC1 has a much less evolutionarily conserved sequence in the 5′ noncoding region compared to GLAST and GLT-1, hindering the identification of Optimal functioning and precise regulation of Glu removal from the synaptic cleft is critical to prevent an excitotoxic insult and thus avoid several neurodegenerative pathologies. To date, compelling evidence suggests that Glu transporters could be regulated in a circadian fashion Figure . It is cDC-C gathered the relevant information, wrote the manuscript, as well as elaborated the figure. AO revised and edited the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Fusarium incarnatum-equiseti species complex (FIESC) comprises 33 phylogenetically distinct species that have been recovered from diverse biological sources, but have been most often isolated from agricultural plants and soils. Collectively, members of FIESC can produce diverse mycotoxins. However, because the species diversity of FIESC has been recognized only recently, the potential of species to cause mycotoxin contamination of crop plants is unclear. In this study, therefore, we used comparative genomics to investigate the distribution of and variation in genes and gene clusters responsible for the synthesis of mycotoxins and other secondary metabolites (SMs) in FIESC.The Fusarium lineages.We examined genomes of 13 members of FIESC that were selected based primarily on their phylogenetic diversity and/or occurrence on crops. The presence and absence of SM biosynthetic gene clusters varied markedly among the genomes. For example, the trichothecene mycotoxin as well as the carotenoid and fusarubin pigment clusters were present in all genomes examined, whereas the enniatin, fusarin, and zearalenone mycotoxin clusters were present in only some genomes. Some clusters exhibited discontinuous patterns of distribution in that their presence and absence was not correlated with the phylogenetic relationships of species. We also found evidence that cluster loss and horizontal gene transfer have contributed to such distribution patterns. For example, a combination of multiple phylogenetic analyses suggest that five NRPS and seven PKS genes were introduced into FIESC from other Our results suggest that although the portion of the genome devoted to SM biosynthesis has remained similar during the evolutionary diversification of FIESC, the ability to produce SMs could be affected by the different distribution of related functional and complete gene clusters.The online version of this article (10.1186/s12864-019-5567-7) contains supplementary material, which is available to authorized users. Fusarium includes some of the most destructive plant pathogens of food and feed crops and produces some of the mycotoxins of greatest concern to food and feed safety. Collectively, Fusarium species (fusaria) are pathogenic on most economically important crops and produce structurally diverse secondary metabolites (SMs), including mycotoxins that have adverse health effects, including immune suppression and cancer , 4529, 4NRPS10 corresponded to the ChNPS10 subfamily; two clades consisting of the fusarin and equisetin PKS-NRPSs subfamily; clades NRPS11, NRPS12 and NRPS13 corresponded to the ChNPS12 subfamily; and clade NRPS22 corresponded to the CYCLO subfamily. For the multimodular NRPSs, clades NRPS1 and NRPS2 corresponded to the SID subfamily, and clades NRPS3, NRPS4, NRPS6, NRPS14, NRPS15, and NRPS17 corresponded to the EAS subfamily.The NRPS analysis included A domains from 269 NRPS genes recovered from the 12 FIESC genome sequences generated in this study and 95 NRPSs from other fusaria. In the resulting tree, the FIESC A domains were resolved into 20 well-supported clades, 15 of which corresponded to NRPS ortholog groups described by Hansen et a1.  NPRS16, NRPS31 or NRPS33 clades to subfamilies. There were some inconsistencies in the phylogenetic relationships inferred in our FIESC A-domain tree compared to those inferred by Bushley and Turgeon [Most of the basal branches in the FIESC A-domain tree did not have significant bootstrap support, and because of this it was not possible to assign the  Turgeon . In the  Turgeon , where t Turgeon  in that  Turgeon  suggest A combination of processes, including gene loss, gene duplication and horizontal gene transfer (HGT), likely contributed to the discontinuous distribution patterns and tree topologies observed for other NRPS/PKS genes. Thus, subsequent analyses were aimed at investigating which of these processes have contributed to the distribution of which NRPS/PKS genes in FIESC.PKS18 was present in all members of the Equiseti clade, and only two members (FIESC 15 and FIESC 25) of the Incarnatum clade. This suggests the gene was present in the common ancestor of the two clades and was subsequently lost during divergence of the Incarnatum clade such that it is currently present only in FIESC 15 and FIESC 25.To assess the contribution of gene loss to the distribution of NRPS/PKS genes in FIESC, we re-examined distribution patterns to determine which NRPS/PKS genes were present in one or more FIESC members but absent in closely related member. We found that six NRPS genes and 10 PKS genes exhibited such distribution patterns Fig. . For exaWe also used NOTUNG analysis to assess loss of NRPS/PKS genes in FIESC. In this analysis, ML trees inferred for each NRPS/PKS gene were reconciled with the species tree shown in Fig. NRPS14, NRPS16, NRPS19, NRPS31 and PKS13 in FIESC  in some FIESC members is also consistent with gene loss events. Within FIESC, we detected pseudogenized versions of ESC Fig. . PKS13 aFusarium lineages  than the corresponding unconstrained trees for 11 of the 17 putative HGT events, including the putative HGT of NRPS22 from the Fujikuroi complex to FIESC  in coding regions of HK genes tend to be positively correlated with divergence of Fusarium species [S values for the transferred gene relative to HK genes in the donor and recipient species, because dS values for the transferred gene should reflect divergence levels since the transfer, whereas dS values of HK genes should reflect divergence levels since the speciation event(s) that led to the donor and recipient. Based on this rationale, we used PAML to generate dS values for each NRPS/PKS gene and for concatenated sequences of 30 HK genes  for a given pairwise comparison of taxa was most often 1.5–4, and even higher for some comparisons  were particularly low (0.27 and 0.30). This suggested that NRPS22 homologs in FIESC23 and FIESC28 began diverging from homologs in the Fujikuroi, Nisikadoi and Oxysporum complexes after FIESC began diverging from these other complexes. Together, results of dS analysis, manual tree comparisons, NOTUNG analysis, and SH-AU tests are consistent with HGT of NRPS22 from the Fujikuroi complex (a close relative of F. nygamai) to a recent ancestor of FIESC 23 and FIESC 25. Putative HGT events for NRPS4, PKS10 and PKS23 were also consistent with results from all the phylogenetic analyses used in this study, whereas other putative HGT events were consistent with results from only one or a subset of analyses . The genes were also present, albeit in a different arrangement, in the genome sequence of Colletotrichum simmondsii (NCBI Accession JFBX00000000.1). The predicted functions of PKS42, NRPS34, and the other seven genes combined with their proximity to one another in multiple taxa suggests that they constitute a SM biosynthetic gene cluster that confers the ability to synthesize a hybrid polyketide-nonribosomal peptide. Examination of the cluster flanking region among members of the Equiseti clade revealed that some of the flanking genes are shared by all members of the clade, suggesting that cluster homologs are in similar genomic contexts  cluster was detected in all FIESC genomes examined in the current study. In seven of the FIESC genome sequences, TRI cluster orthologs were identical to the cluster ortholog previously reported in FIESC 12 (NRRL 13405) [Equiseti clade all genomes examined have an intact and apparently functional TRI1 gene except for FIESC 33 (ITEM 10395). The TRI1 coding region in 10,395 has a single nucleotide deletion near the 5′ end of the coding region that introduces a frameshift and subsequently internal stop codons.The trichothecene biosynthetic gene (L 13405)  with resIncarnatum clade, FIESC 15 strain has an intact and apparently functional TRI1, while TRI1 is absent in the FIESC 23 and FIESC 25 strains and it is pseudogenized in FIESC 28 and FIESC 29. In FIESC 28 there is only a 127 base fragment of TRI1, corresponding to bases 6–132 of the TRI1 coding region, while FIESC 29 has a 122 base deletion near the 5′ end of the TRI1 coding region.These stop codons should prevent translation of a full length Tri1 protein. Within the TRI16 is not located in the TRI cluster, and it is pseudogenized or absent in some species [TRI16 was present and in the same genomic context in FIESC 15 and in all members of the Equiseti clade examined, but that it was absent in F. camptoceras and all other members of the Incarnatum clade.In all fusaria that have been examined,  species , 53, 54.Equiseti or Incarnatum clade, or a few species in both clades. The fusarin biosynthetic gene cluster was among those with discontinuous distributions. As far as we are aware, fusarin production has never been reported in members of FIESC. Therefore, finding the fusarin cluster in three FIESC genomes attests to the value of whole genome sequence analysis to determine the genetic potential of fungi to produce metabolites.The results of this study revealed that collectively members of FIESC have the potential to produce diverse SMs. This was evident by the presence of 22 PKS and 18 NRPS genes in the FIESC genome sequences analyzed. Homologs from approximately 50% of the PKS and NRPS ortholog groups were present in all FIESC genomes examined , F. aywerte, and C. simmondsii , and its absence in FIESC 5 ITEM 11348, indicates two possible evolutionary scenarios for the cluster in FIESC 5: 1) the cluster was present in the common ancestor of CS3069 and ITEM 11348, and was subsequently lost during divergence of ITEM 11348; or 2) the genomic region that includes the PKS42-NRPS34 cluster exists as two alternative alleles, one allele with the cluster and another without it. There is evidence for the existence of similar scenarios with the fumonisin and ochratoxin biosynthetic gene clusters in species of Aspergillus [Aspergillus, our efforts to find evidence for or against the two scenarios for the PKS42-NRPS34 cluster in FIESC 5 yielded equivocal results. For example, we searched for remnants of PKS42-NRPS34 cluster genes in the ITEM 11348 genome sequence as evidence for recent deletion of the cluster, but without any success. Analysis of the PKS42-NRPS34 cluster region in the five haplotypes of FIESC 5 already described [Our analysis showed the presence of a putative cluster, including the ergillus , 56. Howescribed  could prEquiseti or Incarnatum clade, while other clusters were unique to one strain, such as NRPS15 and NRPS17. The antiSMASH prediction of SM clusters is mainly based on PKS, NRPS and terpene synthase (TS) genes, therefore it was not surprising that the predicted gene clusters exhibited the same distribution patterns as PKS and NRPS genes.In this study, we used antiSMASH to estimate the percentage of genes in a genome that are involved in SM biosynthesis Table . While sFusarium species complexes, is the combinations of clusters that are present within each lineage. For example, all members of the FSAMSC that have been examined have the aurofusarin, fusarubin and trichohecene clusters, but not the bikaverin cluster; all members of FFSC that have been examined have the bikaverin and fusarubin clusters but not the aurofusarin or trichothecene cluster; and all members of FIESC examined in the current study have the fusarubin and trichothecene clusters but not the aurofusarin or bikaverin cluster [Fusarium mycotoxin  that occurred in all members of FIESC. In contrast, the enniatin/beauvericin and zearalenone clusters exhibited discontinuous distributions among the genomes , respectively [F. camptoceras, the absence of TRI13 and pseudogenization of TRI7 suggests that this species would produce trichothecene analogs that lack hydroxyl or acetyl substituent at C4. Within the F. sambucinum species complex (FSAMSC), there is a lineage of closely related species in which TRI13 and TRI7 are pseudogenized. As a result, these fusaria produce only trichothecene analogs that lack modifications at C4, including deoxynivalenol (DON), 3-acetyl DON (3ADON) and 15-acetyl DON (15ADON) [F. graminearum, TRI7 and TRI13 are present as pseudogenes and as a result these strains produce either 3ADON or 15ADON. However, most species within FSAMSC have functional TRI13 and TRI7 orthologs. The results of the current study indicate that a similar situation occurs also in FIESC. That is, with the exception of F. camptoceras, FIESC members have functional copies of TRI7 and TRI13 and, therefore, the genetic potential to produce trichothecenes with C4 modifications. Because most members of FIESC and FSAMSC have functional copies of TRI7 and TRI13, we propose that the loss/pseudogenization of TRI7 and TRI13 in F. camptoceras occurred independently of the pseudogenization in FSAMSC. If this hypothesis is correct, it would constitute convergent evolution of the trichothecene biosynthetic pathway in FSAMSC and FIESC. Furthermore, the loss/pseudogenization of TRI7 and TRI13 in two lineages of Fusarium suggests that a lack of selection for production of trichothecenes with a C4 modification has occurred twice during the evolutionary history of trichothecene-producing fusaria.During trichothecene biosynthesis, the ectively . Althoug(15ADON) , 61. ForTRI16 is not located within the TRI core cluster, and it is pseudogenized or absent in some species [TRI16-encoded acyltransferase catalyzes esterification of a five-carbon metabolite (3-methylbutanoate) to a hydroxyl at C8, resulting in the formation of T-2 toxin and structurally related trichothecenes [TRI16 in some members of FIESC, multiple survey studies showed that members of FIESC do not produce T-2 toxin or other trichothecenes with a 3-methylbutanoate ester at C8 [TRI16 homologs in FIESC species are not expressed, not functional due to changes in its amino acid sequence, or that they have taken on a function in a different metabolic pathway. There is evidence that, during the evolutionary divergence of FIESC from other trichothecenes-producing lineages of Fusarium, TRI1 was translocated into the TRI cluster from elsewhere in the genome [TRI1 in TRI cluster orthologs of some members of FIESC suggests that following this translocation, TRI1 was lost in some FIESC lineages   of the low dS ratios for NRPS16, NRPS19, NRPS34, PKS42, PKS43, and PKS69 is not clear.dS ratios also resulted from some pairwise comparisons that involved two strains of the same species  or closely related species . However, in some other comparisons involving strains of the same species or closely related species, dS ratios were high. dS values for both HK and NRPS/PKS genes in comparisons within species or between closely related species tended to be low , and as a result small differences in dS values for HK and NRPS genes could lead to very low or very high dS ratios.Low dPKS23 and NRPS4 genes, respectively. These data provide evidence that a complex interplay of evolutionary processes contributes to variation in secondary metabolite cluster content in fungi and, therefore, to variation in their ability to produce the corresponding metabolites.Overall, all evolutionary approaches hereby considered, showed that HGT has contributed to distribution of four clusters, such as the known fusarin and enniatin/beauvericin biosynthetic gene clusters and the unknown clusters with The results of this study suggest that: 1) collectively, the FIESC strains examined in this study have the genetic potential to produce 22 structurally distinct PKSs and 18 NRPSs; 2) vertical inheritance has contributed to the distribution of almost all FIESC clusters; 3) HGT has contributed to distribution of four clusters and likely to other nine additional clusters, although the results from all analyses were not consistent.Environmental factors that affect the maintenance of SM clusters in a recipient of HGT are poorly understood, but they could include soil conditions, competitors, hosts, and/or climate. However, which ones among these factors affected maintenance of SM clusters horizontally transferred to members of FIESC remains to be determined.In conclusion, all those data can led to understanding which members of FIESC can produce which mycotoxins, concretely impacting growers response to fungal surveys in their fields, and supporting regulators to develop accurate assessments of the risks that members of FIESC pose to the food and feed supply.http://server.ispa.cnr.it/ITEM/Collection) at the Institute of Science of Food production ; two strains were obtained from the U.S. Department of Agriculture (USDA), Agricultural Research Service (ARS) Culture Collection , and one strain was obtained from the Fusarium Research Center . Recently, Villani et al. [Fusarium species were obtained from GenBank for inferring species phylogeny, as well as PKS and NRPS analyses.The FIESC strains examined in this study are listed in Table i et al.  charactei et al. . In the i et al.  as FIESCi et al. . We compi et al. . In addiF. scirpi NRRL 66328, which was generated using a Life Technologies Ion Torrent PGM™ sequencer. Both sequencers were located at the Mycotoxin Prevention and Applied Mycology Research Unit at USDA ARS, Peoria IL. High-quality genomic DNA was prepared from each species grown in GYP  liquid cultures using Zymo DNA Clean & ConcentratorTM-5 kit as described by the manufacture . One ng of DNA was used to generate paired-end DNA libraries for the MiSeq using the Nextera XT DNA Library Preparation Kit . DNA (1 μg) from F. scirpi was used to generate the DNA library for the Ion Torrent using the New England Biolabs NEBnext Fast DNA Library prep set. Sequence reads were processed and assembled with CLC Genomics Workbench version 8.0  using default parameters, except that the minimum contig length was set to 500 bp. The resulting unannotated genome sequences were deposited at DDBJ/ENA/GenBank under the accessions shown in Table F. graminearum as the training species. When we identified predicted gene models that appeared to have errors , we subjected the corresponding genomic sequence to FGENESH [Genome sequences for FIESC strains were generated using an Illumina MiSeq sequencer. The exception to this was the sequence for  FGENESH Fusarium species that spanned the phylogenetic breadth of the genus and for which genome sequence data were publicly available  are not essential, but occur widely as single-copy genes in Fusarium. All DNA sequences of coding regions were inspected, and when necessary were manually annotated to correct large gaps resulting from incorrect prediction of introns and translational start and stop sites.A species phylogeny was inferred using coding region sequences of 30 HK genes − 05) in BLAST searches at NCBI. Sequences were aligned using Mafft [Gallus gallus serving as the outgroup. To gain further insight into diversity of NRPSs in FIESC, we assessed the phylogenetic relationships of A domains of FIESC NRPSs using the same phylogenomic approach that Bushley and Turgeon [Fusarium NRPSs [We used full-length, predicted amino acid sequences to assess diversity and phylogenetic relationships of FIESC PKSs. The PKS analysis included sequences of 135 PKS enzymes identified in the 12 FIESC genome sequences generated during the course of this study, 11 PKSs from CS3069, and 216 PKSs from other fusaria previously identified  or identng Mafft  and an Mng Mafft  with the Turgeon  used to um NRPSs .Fusarium lineages; 2) manual comparison of single NRSP/PKS gene trees to the species tree; 3) constraint analyses using the Shimodaira-Hasegawa and Approximately Unbiased (SH-AU) tests to assess statistical support for branches indicative of HGT [We did a series of phylogenetic analyses to assess evolutionary processes that have likely contributed to the introduction of SM gene clusters into FIESC and subsequent distribution of the clusters among FIESC members. The phylogenetic analyses consisted of: 1) assessment of the occurrence of the genes in FIESC and other e of HGT , 49; 4) e of HGT , 78; ande of HGT  Additional file 2:Fusarium species tree inferred by maximum likelihood analysis of concatenated sequences of 30 housekeeping genes Additional file 4:A. – D. correspond to the major groups of PKS genes previously described in Fusarium and other fungi [A.) and the three subgroups of reducing PKSs: R-PKS I (B.), R-PKS II (C.) and R-PKS III (D.). For each major group, phylogenetically distinct clades (or homolog groups) are labeled with clade numbers  that were previously described in analysis of other fusaria (Brown and Proctor 2016). Homologs from other fusaria were included in current study and are designated using the same abbreviations used by Brown and Proctor [Trees inferred by maximum likelihood analysis of alignment of predicted amino acid sequences of the four major groups of PKS genes retrieved from FIESC genome sequences examined in this study. Supplementary Figs. er fungi . The maj Proctor . NumbersAdditional file 5:Trees inferred by ML analysis of individual NRPS and PKS genes retrieved from FIESC genome sequences examined in this study. All the homologous genes retrieved from NCBI and were included in the analysis. Numbers near branches are bootstrap values based on 1000 pseudoreplicates. Bootstrap values below 70% are not shown. (PPTX 671 kb)Additional file 6:Reconciled NRPS and PKS trees with duplication, losses and HGT events as obtained from NOTUNG. Duplications are indicated with red D, transfers are indicated by yellow T’s, and losses in grey. (PPTX 303 kb)Additional file 7:Fusarium. (DOCX 21 kb)Results of Shimodaira-Hasegawa (SH) and Approximately Unbiassed (AU) tests used to assess branches that suggest horizontal gene transfer of NRPS and PKS genes between FIESC and other lineages of Additional file 8:S analyses. (XLSX 1391 kb)Results of the dAdditional file 9:A. Analysis of homologs of the putative PKS42-NRPS34 cluster. On the left, tree inferred by maximum likelihood analysis of concatenated sequences of the nine genes in the PKS43-NRPS34 cluster. Numbers near branches are bootstrap values based on 1000 pseudoreplicates. Right, gene organization in PKS42-NRPS34 cluster homologs. The vertical dashed lines between genes in the F. aywerte homolog indicate that the genes are one different contigs. The genes on either side of a vertical lines are at the ends of contigs and, therefore, could be adjacent to one another in the genome. B. Analysis of genes flanking the putative PKS42-NRPS34 cluster and their homologs in selected members of FIESC that lack the cluster. The tree to the left was inferred by maximum likelihood analysis of flanking gene F2. Numbers near branches are bootstrap values based on 1000 pseudoreplicates. Bootstrap values below 70% are not shown. Genes and their direction of transcription are represented by arrows. Gray arrows represent genes in the PKS42-NRPS34 cluster, white arrows represent cluster flanking genes, and the yellow arrow represents a gene that was unique to the region in F. camptoceras. (PPTX 154 kb)


Lung transplantation can improve quality of life and prolong survival for individuals with end-stage lung disease, and many advances in the realms of both basic science and clinical research aspects of lung transplantation have emerged over the past few decades. However, many challenges must yet be overcome to increase post-transplant survival. These include successfully bridging patients to transplant, expanding the lung donor pool, inducing tolerance, and preventing a myriad of post-transplant complications that include primary graft dysfunction, forms of cellular and antibody-mediated rejection, chronic lung allograft dysfunction, and infections. The goal of this manuscript is to review salient recent and evolving advances in the field of lung transplantation. Lung transplantation has become an accepted treatment option for patients with various forms of advanced, progressive lung disease that does not respond to non-transplant therapies. Although early post-transplant survival has gradually improved with the implementation of advances in surgical techniques and peri-operative management, many early complications, such as surgical complications or primary graft dysfunction (PGD), threaten lung allograft function and viability, and various complications with delayed onset, such as chronic lung allograft dysfunction (CLAD) or opportunistic infection, continue to significantly impact recipient survival and long-term outcomes. Although an increasing number of patients considered for lung transplantation are extremely ill and require ventilator and/or extracorporeal life support (ECLS) as a bridge to transplant, the last 10 years have seen developments in management pre- and post-transplant that have yielded modestly improved outcomes, including the establishment of revised guidelines for selecting candidates, enhanced surgical techniques, improved methods for donor lung preservation, advances in suppressing and treating allograft rejection, the development of prophylaxis protocols to decrease the risk of opportunistic infection, implementing more effective therapies for treating infectious complications, and the development of a clinical practice guideline concerning the diagnosis and treatment of bronchiolitis obliterans syndrome (BOS) and other forms of CLAD.et al.1 in 2015 under the auspices of the International Society for Heart and Lung Transplantation (ISHLT). A notable change in the eligibility criteria was the recommendation that age by itself should not be considered a contraindication to transplantation with the caveat that patients over 75 years old are unlikely to be candidates, and age over 65 years, if associated with low physiologic reserve and/or other relative contraindications, should remain a relative contraindication. In addition to providing absolute and relative contraindications to lung transplantation, recommendations concerning disease-specific timing of referral and waiting list placement, the use of mechanical and/or ventilator support as a bridge to transplant, special surgical considerations, and other issues were discussed in the document. Of note, the issues of sarcopenia and frailty, which evolving literature has identified as having an important impact on lung transplant outcomes4, were not recognized in the guideline as relative contraindications.A consensus update to previously published documents that addressed the selection of lung transplant candidates was published by Weill6. However, outcomes have been reported to remain somewhat worse for recipients with very high LAS scores9. Nonetheless, it was recently suggested that recipients with higher LAS values and specific transplant indications, such as idiopathic pulmonary fibrosis (IPF) or cystic fibrosis (CF), benefit the most from lung transplantation in terms of survival10, and recipients with the transplant indication of pulmonary fibrosis and very high LAS values can achieve prolonged post-transplant survival that does not differ significantly from that seen in those with lower LAS values11. A recent trends analysis that used the United Network for Organ Sharing (UNOS) database found that recipients with LAS values in the upper quartile transplanted in the more recent era of 1 January 2012 to 31 March 2014 had significantly improved mortality at 30 days and 1 year versus those in earlier eras12. The success of the LAS system in the US has led to its adoption in non-US countries (Germany in 2011 and the Netherlands in 2014)13. Nonetheless, there remains a need to further improve the lung allocation process to decrease the risk of poor outcomes and associated increased costs.The lung allocation score (LAS) was implemented in 2005 to provide a system for donor lung allocation in the US based on urgency and benefit rather than time accrued on the waiting list, and use of the LAS has reduced the risk of death without transplant for waitlisted patients while improving the effectiveness of organ allocation and transplantation by increasing the number of transplants and shifting the distribution of donor lungs to patients who are more likely to die on the waiting list14. Another source of donor lungs that is increasingly being used to help meet the persistent shortage of donor lungs is that of donation after circulatory death (DCD) donors, a source that remains considerably underutilized but could significantly increase the pool of donor lungs, especially when coupled with donor lung resuscitation and evaluation usingex vivo lung perfusion (EVLP). Chancelloret al.15 performed a chart review at their quaternary care center and identified 85 patients (out of 512 who did not have any evidence of lung pathology) over a one-year time period who met criteria that qualified them as potential DCD lung donors. Costaet al.16 compared outcomes for 46 DCD lung transplants (lungs were suitable for use from 44 out of 73 DCD donors) to those of 237 transplants from DBD donors and found that rates of PGD and survival were equivalent for DCD versus DBD transplants. Similarly, a multicenter study performed in the Netherlands did not identify significant differences in PGD, freedom from CLAD, or overall survival for 130 DCD recipients versus a matched cohort of 130 DBD lung recipients17, and Ruttenset al.18 retrospectively reported a similar experience from Belgium in which no significant differences in time of ventilator support, length of intensive care unit or hospital stay, PGD score, freedom from CLAD, or overall survival for 59 DCD as compared to 331 DBD recipients were identified.Donation after brain death (DBD) donors represent the traditional and largest source of donor lungs, and relaxing ideal criteria to allow the use of marginal donor organs that meet extended criteria has increased the numbers of lungs available for transplant20. However, the use of ECLS as a bridge to transplant for patients with severe respiratory failure has gradually increased23, and ECLS may provide the only means of keeping a patient alive and can also be used to support recipients through the transplant procedure27. An evolving experience with ambulatory ECMO has demonstrated that this approach can be used to avoid deconditioning and allow patients to be ambulatory while awaiting organ offers and transplantation32, and using ambulatory ECMO has recently been shown to have economic advantages over non-ambulatory ECMO33. Recent queries of the UNOS database have shown that while the use of ECMO had an adverse impact on survival for older patients, a negative effect was not demonstrated for a subgroup of younger patients under 40 years of age34, and when used in high-volume transplant centers, ECMO use did not appear to increase the risk of post-transplant mortality35.Outcomes for lung recipients on either mechanical ventilation or devices that provide ECLS pre-transplant are generally worse than outcomes for recipients who do not require such support37. While donor lungs are supported by EVLP, the physiologic status of lungs with marginal function and/or evidence of significant injury can be observed and potentially improved42, and if an unacceptable degree of persistent dysfunction is identified, transplantation of inadequately functioning donor lungs can be avoided. EVLP allows fluid to be drawn from extravascular compartments in edematous lungs such that gas exchange can be improved and marginal lungs rendered usable, and anti-inflammatory cytokines or mesenchymal stem cells (MSCs) can be employed to accelerate repair after injury, encourage recovery in intercellular alveolar epithelial tight junctions, improve oxygenation, and decrease vascular resistance46. In addition, antibiotics can be administered to reduce or eradicate donor lung infection47, gastric acid-injured donor lungs can be potentially salvaged48, and perfusate can be analyzed to detect biomarkers that predict high risk for early allograft dysfunction50. Finally, Yeunget al.51 recently demonstrated that acellular normothermic EVLP was associated with a progressive decline in cell-specific leukocyte gene expression over a 12-hour period, which suggested that this technique can potentially interrupt an inflammatory response via washout of intravascular leukocytes, and metabolomic characteristics of EVLP perfusate may serve as biomarkers that can predict PGD risk and improve the selection of marginal lungs that can be used for transplant49. EVLP is likely to significantly expand the usable donor pool as donor lungs are provided to both large- and smaller-volume institutions from EVLP hubs56. Two EVLP devices have been approved by the Food and Drug Administration for clinical use, and a number of clinical trials evaluating the use of EVLP to evaluate and potentially recondition marginal donor lungs are currently in progress.EVLP allows explanted donor lungs to be perfused and ventilated while being evaluated and reconditioned prior to implantation2 /FiO2  ratios with >300 for mild (stage 1), 200–300 for moderate (stage 2), and <200 for severe (stage 3)57. Notably, analysis of data from the Lung Transplant Outcomes Group multicenter recipient cohort  did not detect mortality differences between patients with no (stage 0) and mild (stage 1) PGD, and the authors concluded that the PGD consensus definition could be simplified by combining stages 0 and 158.PGD is defined as lung injury that occurs within the first 72 hours following lung implantation as reflected by the appearance of diffuse allograft edema/infiltration on chest radiograph, and PGD severity is staged according to PaO57, and while a number of markers have been identified that correlate with increased risk of high-stage PGD60, interventions other than supportive care have been relatively ineffective in preventing or treating PGD62. Risk factors for developing PGD include both donor- and recipient-related factors, surgical factors at the time of transplantation, epithelial injury, endothelial injury and activation, and triggering of both innate and adaptive immune responses60. A key early event in PGD pathogenesis is the translocation of neutrophils from circulating blood to the interstitium and air spaces in response to chemotaxins such as damage-associated molecular pattern (DAMP) moieties or CXCL8 that are released as a consequence of ischemia-reperfusion injury61. Various pro-inflammatory cytokines, reactive oxygen intermediates, and proteolytic enzymes represent prominent mediators of tissue disruption in the reperfused, ischemia-damaged lung, and other cell types, such as lung macrophages and lymphocytes, also play a role in the acute lung injury and the loss of epithelial integrity and resultant interstitial edema that occurs in PGD.Nearly one-third of lung transplant recipients have been reported to develop stage 3 PGD62. Other recent studies have identified a pathogenic role of neutrophil extracellular traps63, a decreased risk of stage 3 PGD when allografts are oversized as reflected by predicted donor and recipient total lung capacity estimations64, variations in oxidant stress gene expression and PGD stage 3 risk65, and a role for cell-free hemoglobin in causing oxidative endothelial injury that can increase vascular permeability and contribute to PGD66.A number of collaborative studies performed and published by the Lung Transplant Outcomes Group have provided additional insights into PGD risk factors and pathogenesis. Notable observations include the association of a genetic variant, Toll-interacting protein rs3168046 (TOLLIP), with clinical PGD and with higher circulating plasminogen activator inhibitor-1 (PAI-1), which has been previously identified as a quantitative PGD lung injury biomarker67. Of interest, it has been shown that in a mouse model involving primary transplantation of fully major histocompatibility complex (MHC)-mismatched lungs that were subjected to a 72-hour period of costimulatory pathway blockade (peri-operative blockade of B7-CD28 and CD40–CD40 ligand costimulatory pathways), retransplantation of the lungs into T-cell-deficient recipients led to long-term acceptance that could be abrogated if the primary recipients had received anti-CD25 antibodies68. Another murine model study by this group identified CCR7-expressing CD8+ T cells that interacted with CD11c+ antigen-presenting cells and infiltrated lung allografts as playing a critical role in tolerance induction69. Although immune checkpoint inhibition has been studied fairly extensively in animal models of allogeneic organ transplantation, experience with immune checkpoint inhibitors to modulate allograft tolerance in human organ transplant recipients is limited to anecdotal case reports70 and a phase I/Ib clinical trial in allogeneic stem cell transplantation in Japan71. Advances in understanding immune tolerance and translating such knowledge to clinical application in allogeneic lung transplant represent a key strategy to lessening the risk of PGD, acute rejection, and CLAD. Simultaneous infusion of donor bone-marrow-derived cells at the time of lung transplantation has been associated with a higher incidence of donor-specific hyporeactivity as determined by one-way mixed leukocyte culture as compared to controls72, and long-term lung allograft survival without immunosuppression in a patient who simultaneously received T- and B-cell-depleted bone marrow has been reported73.A key goal in lung transplantation is to achieve a state wherein a destructive alloimmune response does not occur when immune responses are not globally suppressed. Mixed chimerism has been shown to allow tolerance to emerge in both murine and large animal models, although mechanisms by which this is achieved differ among species, and sustained chimerism and tolerance have been particularly difficult to achieve in primates76. This syndrome was initially termed bronchiolitis obliterans syndrome (BOS) in the 1990s78 and diagnosed on the basis of a persistent ≥20% decline from best post-transplant forced expiratory volume in one second (FEV1) without other identifiable cause, and BOS was assumed to be a consequence of chronic rejection and the cause of persistent decline in the surrogate marker of FEV1. It subsequently became clear that a multitude of complications can cause such a decline in FEV174, and the term CLAD has supplanted the formerly used, overarching term BOS79. Recent analyses of the literature and a consensus revision of terminology have recognized two major CLAD phenotypes, which are obstructive CLAD (BOS) and restrictive CLAD 79. The American Thoracic Society (ATS)/European Respiratory Society (ERS)/ISHLT clinical practice guideline systematically examined available evidence that had accrued in the literature pertaining to BOS/CLAD, and an expert consensus committee provided recommendations for its diagnosis, prevention, and treatment74. Identified risk factors included PGD, various forms of alloimmune rejection , infections , pathologic GER, autoimmunity, and persistent bronchoalveolar lavage (BAL) neutrophilia. Despite the evidence from randomized controlled trials (RCTs) in the prevention and treatment of BOS/CLAD being low or very low in quality, a number of conditional recommendations were made by consensus among task force members following a comprehensive review of available publications  combined with established immunosuppressive regimens, but other approaches  had few data to support their therapeutic use.Restrictive CLAD has a worse prognosis than does obstructive CLADet al.86 examined outcomes data from French and Swiss transplant cohorts (SysCLAD Consortium) and reported that the best prediction model for early onset CLAD included underlying diagnosis as the indication for transplant, type of induction regimen, maintenance immunosuppression regimen, and whether donor-specific antibodies (DSA) directed against class II HLAs were present at one year post-transplant. Many biomarkers have been associated with CLAD, but none have been validated as clinically useful. Specket al.87 performed a comprehensive literature search that sought to identify potentially useful cytokines  in plasma and/or BAL, but none could be considered suitable as diagnostic markers. More recently, Durandet al.88 reported that a threshold increase in CD4+CD25hiFoxP3+ T cells at one and six months post-transplant was associated with a two-fold increased risk of developing BOS.Predicting risk for developing CLAD has been elusive. Koutsokera89. Hayneset al.90 evaluated whether a HLA-DR restricted effect on BOS/CLAD incidence could be detected in a large, single-center recipient cohort with long-term follow up; the presence of donor HLA-DR15 (which avidly binds α1 chain peptides of collagen V [colV] and is associated with anti-colV autoimmunity) was associated with a significant increase in susceptibility to severe BOS, while donor HLA-DR7 or recipient HLA-DR17 expression was associated with reduced susceptibility.Additional recent publications of interest include those examining the potential utility of magnetic resonance imaging as an adjunct to spirometric measurement of FEV1 to diagnose CLAD and characterize/quantitate CLAD-related change92. Bacteria, archaea, fungi, and viruses that comprise specific lung microbiomes are constantly interacting with each other and with host immune responses and defense mechanisms, and lung transplant recipients must adapt to the newly acquired donor microbiome94. Sharmaet al.95 have reported that microbiome signatures of the mouth, nose, and proximal and distal airways in transplant recipients show site-dependent variation, with the nasal microbiome closely resembling that of the proximal and distal airways; myeloid-derived suppressor cell (MDSC) phenotypes were found to predominate in proximal airways, while a preponderance of pro-inflammatory MDSCs was found in distal airways, and the abundance of distinct bacterial phyla correlated with specific MDSCs in proximal versus distal airways. Mourauxet al.96 demonstrated a correlation between the presence of pro-inflammatory bacteria  in BAL and catabolic lung remodeling gene expression; however, when bacteria known to be associated with a healthy, steady-state microbiome  were found in BAL, so was the expression of anabolic remodeling genes. Additional investigation of the transplanted lung microbiome and host–microbiome interactions is needed to provide insights into injury responses and matrix remodeling as well as alloimmune responses, allograft tolerance, and the pathogenesis of CLAD94.Investigations of the lung microbiome in health and disease have shown that it varies considerably among disease states99, MSCs may also be capable of inhibiting immune cell function  and cytokine secretion101. Numerous studies in animal models have demonstrated stem cells’ ability to lessen injury and inflammation103, and stem cell therapies may blunt acute ischemia-reperfusion injury and fibrotic responses to lung injury, thereby preventing or limiting the severity of PGD103. In addition, MSCs may have the capacity to encourage alveolar fluid clearance in lungs that otherwise would not be suitable for transplantation because of edema and impaired gas exchange104, and early experience using allogeneic bone-marrow-derived MSCs in human lung transplant recipients with deteriorating CLAD showed slowing of the decline in FEV1 and supports the feasibility of using such therapies in patients with advanced CLAD105.Although donor-derived MSCs have been identified as potentially playing a significant role in bronchiolar fibrosis in BOS/CLAD107. Extensive genetic engineering of pigs as a lung donor source is ongoing, but genomic restructuring is needed for the prevention of the acute thrombotic and severe inflammatory reactions seen in various models such as pig and primate xenotransplantation109. Ongoing efforts to knock out xenoantigens to avoid triggering coagulation and inflammatory cascades and complement activation in partnership with pharmacologic manipulation could one day lead to the transplantation of pig lungs to humans110.Xenotransplantation could be an alternative approach to allogeneic lung transplantation or act as a bridge to allotransplantation by extending the time period for a human donor–recipient match to be found with subsequent replacement of the xenograft when a human donor lung becomes available112. Research is ongoing to determine whether bioengineered lungs that have been adequately re-cellularized with appropriately differentiating stem cells that can replace or repopulate the over 40 various cell types in their usual anatomic sites can sustain prolonged function113.Although bioartificial lung grafts derived by re-cellularizing an intact whole lung extracellular matrix with stem or progenitor cells have been successfully placed orthotopically in animal models and can provide gas exchange, delayed onset of inflammation and consolidation has eventually led to loss of functionMany key advances  have occ


Candida antarctica lipase B, and their application to create new functionalized polymers, including the regio- and chemoselectivity of the reactions.Enzyme catalyzed reactions are green alternative approaches to functionalize polymers compared to conventional methods. This technique is especially advantageous due to the high selectivity, high efficiency, milder reaction conditions, and recyclability of enzymes. Selected reactions can be conducted under solventless conditions without the application of metal catalysts. Hence this process is becoming more recognized in the arena of biomedical applications, as the toxicity created by solvents and metal catalyst residues can be completely avoided. In this review we will discuss fundamental aspects of chemical reactions biocatalyzed by  The field has received widespread interest in the past decade due to its ability to harness chemical innovation to simultaneously meet environmental and economic goals . SynthesEnzymes are Nature’s catalysts that accelerate specific metabolic reactions in living cells. As an environmentally friendly alternative to conventional chemical catalysts, enzymes offer several advantages, including high selectivity, high efficiency, the ability to operate under mild conditions and catalyst recyclability. Answers to the question “Why use enzymes?” include necessity, convenience and opportunity. New synthetic and catalytic methods are necessary to design new classes of functional polymers for covalent polymer-drug conjugates. Despite all the advantages that enzymatic catalysis offers, the area of quantitative functionalization of preformed polymers has not fully been developed. Enzymes can catalyze the modification of a polymer through functional groups located in the main chain, in the side chains or at the polymer terminals (synthetic symmetric or asymmetric telechelic polymers). Enzymes are classified into six main groups according to the International Union of Biochemistry and Molecular Biology  6]. Tod. Tod6]. in vivo. They are widely used in esterification, transesterification, aminolysis, and Michael addition reactions in organic solvents ..6].The catalytic cycle of the CALB-catalyzed transesterification of vinyl acetate (VA) with 2-phenylpropane-1-ol (PPOH) is visualized in Although an increase in ester concentration was shown to increase the rate and conversion of transesterification ,27, it wThe structure of the alcohol is also an important parameter affecting the rate and overall conversions in enzymatic transesterification. It was observed that straight-chain alcohols gave better conversion compared to aromatic and branched-chain alcohols in the CALB-catalyzed transesterification of vinyl acetate due to less steric hindrance around the hydroxyl group  24]. Ho. Ho24]. 1H-NMR spectrum shows that the product obtained constitute one thiol end group and one vinyl end group which confirms only one vinyl group of DVA participated in the transesterification reaction , , a comb-like polymer, and its corresponding monomer were acylated with vinyl acetate, phenyl acetate, 4-fluorophenyl acetate and phenyl stearate in THF in the presence of Pseudomonas fluorescens. The copolymer was acylated with about 40% conversion after 7 days when phenyl acetate was used as the acyl donor [Polyphenol oxidase catalyst, Only 1.3% of aniline was incorporated [The -OH groups in the side chains of poly[yl donor . Poly can selectively epoxidize polybutadiene (nM = 1300 g/mol)  in organic solvents in the presence of CALB 2Si-CH2-OH) [-CH2-OH) ,71 was qIn terms of CALB-based transformations in solution: Challenges surround issues of incompatibility between the solvents tolerate by the CALB and the substrates solubility. In the case of solventless transformations, the substrate must be liquid at the working temperature (50 °C) and in the case of polycondensation the formation of highly viscous product limits the application. If the reaction proceeds for long periods of time, the CALB starts to catalyze the hydrolysis of the polyesters. As a result, it is still quite challenging to synthesize high molar mass polyesters. The control of polymerization in this case remains less efficient with enzymes than with chemical initiators.Enzyme mediated functionalization of polymers can be used to generate new functionalized polymers. Chemical reactions specifically transesterification, Michael addition, ring opening polymerization can be conducted at milder conditions exploiting the catalytic activity of CALB. This method is green and can be used to synthesize polymers for biomedical applications as many cases no solvent and no metal catalysts were used. Enzymes also offer region- and chemoselectivity not achievable using conventional catalysis.


Acute myeloid leukemia (AML) is a genetically heterogeneous malignancy for which treatment options have been largely limited to cytotoxic chemotherapy for the past four decades. Next-generation sequencing and other approaches have identified a spectrum of genomic and epigenomic alterations that contribute to AML initiation and maintenance. The key role of epigenetic modifiers and the reversibility of epigenetic changes have paved the way for evaluation of a new set of drug targets, and facilitated the design of novel candidate treatment strategies. More recently, seven new targeted therapies have been FDA-approved demonstrating successful implementation of the past decades' research. In this review, we will summarize the most recent advances in targeted therapeutics designed for a focused group of key epigenetic regulators in AML, outline their mechanism of action and their current status in clinical development. Furthermore, we will discuss promising new approaches for epigenetic targeted treatment in AML which are currently being tested in pre-clinical trials. Epigenetic regulation of the state of a cell involves various dynamic and reversible post-translational modifications of DNA and histone proteins. These modifications in their totality regulate the accessibility of DNA for the transcription machinery, thereby determining which specific genomic loci are transcriptionally active or repressed . The besFLT3 and KIT), transcription factors (such as CEBPA and RUNX1), chromatin modifiers (such as MLL and ASXL1) or direct and indirect regulators of DNA methylation  , The Cancer Genome Atlas TCGA, 2006), the International Cancer Genome Consortium ICGC, 2008), and the European Community initiative BLUEPRINT (2011) have been critical in defining the regulatory networks in different normal hematopoietic cell types as well as how they are deregulated in myeloid malignancies 003, The . The key, the Intnd TET2) , 8. Alth, and the+ AML alone or in combination with conventional chemotherapy; the multi-kinase inhibitor midostaurin for newly diagnosed FLT3-mutated AML in combination with conventional chemotherapy; and the FLT3, AXL, and ALK inhibitor gilteritinib for relapsed or refractory FLT3-mutated AML  and arsenic trioxide (ATO) for treatment of acute promyelocytic leukemia  and the ated AML –19. IDH1ated AML . As a geated AML  and so rWhile these new developments in FDA approval are welcome, there remains much to do to improve the outcome of patients with myeloid malignancies. In this perspective, we will discuss a discrete set of candidate epigenetic therapeutic targets currently under evaluation in AML: the lysine demethylase LSD1, the protein methyltransferases EZH2, DOT1L, and PRMT5, and the BET bromodomain proteins. We will describe the importance of these transcriptional activators and repressors in different AML subtypes as well as their targeting potential, possible limitations and potential toxicities. We will summarize their current status in clinical development. For detailed review of other equally important targets, such as DNMTs and HDACs the reader is referred to recent comprehensive reviews –22. FinaHistone methylation and demethylation are tightly regulated, dynamic processes that regulate transcriptional activation or repression depending on the location of the modification. Methylation is generated by specific histone methyltransferases (HMTs), such as MLL, DOT1L, and EZH2. As for other histone modifications, methylation can be reversed by two classes of demethylases (KDM): the larger Jumonji domain family and the smaller lysine-specific demethylase (LSD) family.LSD1/KDM1A is a flavin-adenine dinucleotide (FAD)-dependent histone demethylase , 24 withMLL gene translocations  and iadademstat , have been evaluated in phase I trials in patients with relapsed or refractory AML . While tocations , 38. PreEZH2 is the catalytic subunit of Polycomb Repressive Complex 2 (PRC2) which is responsible for maintaining transcriptional repression of its target genes through tri-methylation of H3 K27 , 40 Fig. This hiMLL-rearranged AML, the dual EZH1–EZH2 inhibitor DS3201b has entered phase 1 as monotherapy in patients with refractory acute leukemia (NCT03110354)  where S-adenosyl methionine-competitive inhibitors, such as GSK2816126, CPI1205, and the most promising, tazemetostat (EPZ-6438), have been evaluated. Another is MAK683 which is an EED-binding complex disrupter under investigation in refractory lymphoma and solid malignancies (NCT02900651). Phase I data for tazemetostat demonstrated an acceptable safety profile and some objective responses (NCT01897571) resulting in initiation of a number of follow on single agent and combination studies . On the basis of pre-clinical studies demonstrating the functional importance of PRC2 in 3110354) . HoweverMLL (KMT2A) occurs in around 5% of AML cases, predominantly resulting in an MLL-AF9 fusion gene, although other partner genes occur less frequently .The S-adenosyl methionine-competitive DOT1L inhibitor pinometostat (EPZ-5676) displays great specificity for DOT1L over other histone methyltransferases –63. Prec in vivo . Phase Iompleted   family of proteins, and was identified as a potential cancer therapeutic based on results of a genome-wide shRNA screening in MLL-dependent AML cells , 89. BRDpression . Geneticenhancer , but alsex vivo patient samples or mouse models, in particular in specific subtypes with MLL rearrangement, or those with mutations in NPM1, FLT3 or IDH2, or EVI1 overexpression  and histone deacetylases (HDACs) catalyze the dynamic and reversible acetylation of histone and non-histone proteins, and are involved in major epigenetic regulatory mechanism of gene transcription  in normaMOZ or MLL in leukemia  has been demonstrated to have potent anti-proliferative and pro-differentiation activity in AML cell lines and primary patient samples  . MEN1 seMEIS1  (Figure MEIS1 . InteresMEIS1 , genetic(in vivo , highlig(in vivo , 116, whHOXA and MEIS gene expression, thereby releasing the differentiation block associated with MLL-rearranged leukemia (MLL translocations (in vivo is still an important hurdle to overcome.Several small molecules and tool compounds targeting the interactions of components of the MLL complex have been recently developed. Inhibitors targeting MEN1-MLL have been shown to reverse leukemia –121. Simocations  or CEBPAocations . HoweverEpigenetic regulators are central players in the initiation and maintenance of hematopoietic malignancies, an observation which has resulted in myriad opportunities for development of targeted therapies. In particular, where epigenetic mechanisms are specifically disordered in malignant but not normal blood cells, there exists the potential for a significant therapeutic window. Along these lines, there has been significant progress in understanding the role of epigenetic modifications and their modifiers in cancer in general and in AML in particular. The discovery and development of small-molecule inhibitors targeting certain epigenetic regulators has already led to opportunities for clinical trial evaluation and potential patient benefit; impending trial results will inform on efficacy and safety. While the evaluation of many of these compounds for their single agent activity is an essential first step, it will be critical to test efficacy in combination with either standard-of-care chemotherapies or novel therapeutics, to determine their optimal role in the treatment of leukemia. In time, personalization of therapeutic regimens according to patient cytogenetics and molecular mutations may become of essential importance. A final point is that the interdependence of cancer epigenetics and immunological responses has to be taken in consideration. In the setting of leukemia, this includes the therapeutic modality of allogeneic stem cell transplantation. It is clear that epigenetic therapies can induce cellular responses in tumor cells that interact with the immune system and which may contribute to their efficacy; for example, effects of adoptive immunotherapies and immune checkpoint inhibitors can be potentiated by epigenetic therapies . As suchBW and TS conceived and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.