Notch and interleukin-22 (IL-22) signaling are known to regulate tissue homeostasis and respond to injury in humans and mice, and the induction of endogenous aryl hydrocarbon receptor (Ahr) ligands through Notch links the two pathways in a hierarchical fashion. However in adults, the species-, organ- and injury-specific gene expression of the Notch-AhR-IL22 axis components is unknown. We therefore performed gene expression profiling of DLL1, DLL3, DLL4, DLK1, DLK2, JAG1, JAG2, Notch1, Notch2, Notch3, Notch4, ADAM17/TNF-α ADAM metalloprotease converting enzyme (TACE), PSEN1, basigin (BSG)/CD147, RBP-J, HES1, HES5, HEY1, HEYL, AHR, ARNT, ARNT2, CYP1A1, CYP24A1, IL-22, IL22RA1, IL22RA2, IL10RB, and STAT3 under homeostatic conditions in ten mature murine and human organs. Additionally, the expression of these genes was assessed in murine models of acute sterile inflammation and progressive fibrosis. We show that there are organ-specific gene expression profiles of the Notch-AhR-IL22 axis in humans and mice. Although there is an overall interspecies congruency, specific differences between human and murine expression signatures do exist. In murine tissues with AHR/ARNT expression CYP1A1 and IL-22 were correlated with HES5 and HEYL expression, while in human tissues no such correlation was found. Notch and AhR signaling are involved in renal inflammation and fibrosis with specific gene expression changes in each model. Despite the presence of all Notch pathway molecules in the kidney and a model-specific induction of Notch ligands, IL-22 was only up-regulated in acute inflammation, but rapidly down-regulated during regeneration. This implies that for targeting injury responses, e.g. via IL-22, species-specific differences, injury type and time points have to be considered. IL-20 gene family [in vivo is regulated by Notch signaling [Tissue homeostasis has been identified as the key process for survival of multicellular organisms by Claude Bernard in the 19th century . Tissue e family ) particue family ,18. Intecis- and trans-signaling mediators, respectively. The in-cis elements, i.e. Notch ligands are members of the δ and the Jagged gene families [trans expressed Notch receptors Notch1–4 [cis Ahr/IL-22 signaling depends on ligand-induced heterodimerization of AHR with either ARNT [p-dioxin (TCDD), but also endogenous tryptophan-derivatives and bilirubin. Upon heterodimerization, AHR/ARNT binds to the so-called xenobiotic response elements (XRE) in the promoters of the Ahr target genes CYP1A1 [CYP24A1 [I-L22 [trans expressed IL22 receptor, which comprises the specific IL22RA1 chain and the common cytokine receptor IL10R2 chain [trans target cell that are mediated mainly by STAT3, which after phosphorylation acts as a transcription factor for Ahr/IL-22 signaling target genes [Both the Notch  and the families . DLL andNotch1–4 , a proceNotch1–4 ,26. AfteNotch1–4  and the Notch1–4  cleave tNotch1–4 . After cNotch1–4  to regulNotch1–4  and HEY Notch1–4  proteinsher ARNT  or ARNT2her ARNT . AHR ligs CYP1A1 , CYP24A1[CYP24A1 , and I-L1 [I-L22 , amongstR2 chain . A solubR2 chain . Finallyet genes .Because of the importance of Notch and Ahr-dependent IL-22 signaling in tissue regeneration upon injury, we speculated on a consistent induction of both pathways upon injury in human and murine tissues.JAG1, NOTCH2, CD147, and STAT3 were most abundantly expressed in all the tissues. Gene expression of pathway molecules was detected in all the tissues, even though expression of HES5 was low in lung and spleen, and DLK1 was low in colon and bone marrow. While myocardial, testicular, and renal tissues showed strongest expression of Notch pathway molecules, the Ahr pathway components showed highest expression in the brain, heart, and kidney. While most organs had high expression of ARNT, which is known to dimerize with Ahr for nuclear signaling, the brain expressed very little ARNT, but had highest expression of ARNT2 amongst all tissues, potentially indicating different Ahr signaling in the brain compared with other tissues.The relative expression of Notch-AhR-IL22 pathway molecules in ten human organs is shown in In the next step, we characterized expression of the same genes in the murine counterparts of the aforementioned organs. The relative expression of Notch-AhR-IL22 pathway molecules in ten murine organs is shown in When we correlated the respective gene expression in humans and mice, we found a significant correlation of expression for all organs with the exception of bone marrow . To bettHes1 and Hes5, which also showed transient up-regulation followed by prolonged down-regulation during the 24 and 48 h of regeneration after injury. Interestingly, this pattern was not observed in the regeneration phase after rIRI, where Hes1, Hes5, Hey1, and HeyL all were significantly induced, with HeyL still being up-regulated 10 days after initial injury  and acute renal oxalate crystallopathy (rAOC) are shown in l injury A. In rAOl injury B. In conAcute inflammation is followed by regeneration to restore tissue homeostasis, but if the injurious trigger persists, irreversible damage of tissues occurs. In the kidney, this leads to epithelial atrophy, which is accompanied by interstitial fibrosis caused by maladaptive repair mechanisms. We therefore next analyzed two mouse models of chronic tubular injury and atrophy. While short exposure to oxalate crystals induces reversible rAOC, long-term oxalate overload induces renal chronic oxalate crystallopathy (rCOC) like in primary hyperoxaluria type 1. Another well characterized model of chronic kidney disease (CKD) is unilateral ureteral obstruction (UUO), in which intraluminal hydrostatic pressure induces the tubular atrophy. Gene expression patterns of the Notch-AhR-IL22 pathway in both models are shown in r² =0.36). While most organs had high expression of ARNT, which is known to dimerize with Ahr for nuclear signaling, the brain expressed very little ARNT, but had highest expression of ARNT2 amongst all the tissues, potentially indicating different Ahr signaling in the brain compared with other tissues. This is particularly interesting, as in rCOC, chronic injury strongly induced ARNT2 along with Ahr expression, probably leading to altered downstream signaling of the Ahr pathway. When we analyzed the correlation between Notch and Ahr/IL-22 target gene expression, the Ahr-dependent ligand expression correlates less with Notch target genes in humans than in mice. As the difference is most pronounced in lung and liver, probably exogenous Ahr ligand from human environments not present in relatively enclosed laboratory environments account for this phenomenon.The Notch and the Ahr/IL-22 signaling pathways are well described and recently, a hierarchical connection of the two pathways has been described ,20. RelaIn the present paper, we not only characterized homeostatic gene expression, but also analyzed expression patterns of the Notch-AhR-IL22 pathway in different models of acute and chronic injury. As Kulkarni et al.  and BielWhile for IL-22, we provide histological evidence for consistent mRNA and protein expression, the data provided here mostly relied on mRNA expression as assessed with RT-qPCR. While we can provide compelling evidence for regulation of specific genes of the Notch-AhR-IL22 pathway, obviously these results should be validated on the protein level. Beyond that, based on the design of our study, we cannot prove any causal role for the genes being identified in the different models. As some of our observations are in agreement with already published roles of single genes , it woulWe procured human total RNA Master panel II (Clontech) and selected an array of human solid organs. The used modified guanidinium thiocyanate method ,46 ensurTwelve weeks old adult C57BL/6 mice were ordered from Charles River, Sulzfeld, Germany. Mice were housed in groups of five in polypropylene cages under specific pathogen-free conditions with free access to food and water. They were kept under standard conditions in a 12-h light/dark cycle. Under general anesthesia, mice were killed by cervical dislocation and tissues were immediately transferred to RNAlater . Ten milligrams of tissue mass was used to obtain high quality, DNA-free, RNA with Pure Link RNA Mini Kit  according to manufacturer’s instructions, to ensure high-quality DNAse solution was applied to each sample and traces of DNAse were eliminated by additional washing steps. NanoDrop 1000 spectrophotometer  was used to assess the concentration and purity of aqueous RNA samples as described ,49. In bn=5) were flank incised under anesthesia and the renal artery of one kidney was clamped for 15, 25, and 35 min. Afterward, kidneys were reperfused for 12, 24 h, 7, and 10 days, a well-established [Groups of 8-week-old C57BL/6 mice . For all quantitative real-time PCR experiments LightCycler 480  was used. Our established protocol [in silico specificity screen (BLAST). Primer design ensured specificity for cDNA transcripts and covered most CCDS-approved transcripts. Primers were designed to amplify an amplicon at a length between 80 and 200 bp. PCR amplification kinetics (efficiency) of each primer were tested prior to experiments with efficiencies between 1.6 and 2. Unspecific binding and dimer formation was minimized by in silico analysis prior to experiments. Additionally, for every sample melting curves profiles were analyzed in order to detect unspecific products and primer dimers and all controls consisting of ddH2O were negative for target and housekeeping genes as expected. The efficiency-corrected quantitation was performed automatically by the LightCycler 480 based on extern standard curves describing the PCR efficiencies of the target and the reference genes (ratio = EtargetΔCPtarget (control − sample)/ErefΔCPref (control − sample)). The high confidence algorithm was applied in order to minimize the risk of false-positive crossing point (Cp). If samples did not rise above the background fluorescence (Cp or quantitation cycle Cq) of 35 cycles during the amplification reaction they were regarded as not detectable, whereas samples with Cp between 5 and 35 cycles were regarded as detectable [The mRNA expression levels of the AhR/Notch/IL-22 axis in human and murine solid organs were assessed by quantitative RT-PCR. Ribosomal 18S rRNA was used to normalize mRNA expression of target genes. In order to control sample homogeneity geometric mean (GM), arithmetic mean (AM) minimal value, maximal value, S.D., variance, and coefficient of variance (CV) of the housekeeping gene 18S were calculated (Supplementary Table S2+ S3). According to established protocols ,56, eachprotocol  comprisitectable .After killing the mice, one middle part of each kidney was directly transferred into 4% neutral-buffered formalin for fixation. After 12 h, the tissues were processed by dehydrating in graded alcohol and by embedding in paraffin for long-term storage. From paraffin blocks, 4-μm sections were generated and further processed by deparaffinizing, rehydrating, and transferring into citrate buffer. Microwave treatment or autoclaving was performed for antigen retrieval by established protocols . For immP<0.05 indicated statistical significance) was used to assess comparison between groups and post hoc Bonferroni’s correction was applied in order to correct multiple comparisons. In order to assess correlation between murine and mouse tissue samples, Pearson’s r was calculated using GraphPad Prism.Data are shown as mean ± S.E.M. Univariate ANOVA (a value of


Arabidopsis thaliana, the required genes for tocopherol biosynthesis and functional roles of tocopherols were elucidated in mutant and transgenic plants. Recent research efforts have led to new outcomes for the vitamin E biosynthetic and related pathways, and new possible alternatives for the biofortification of important crops have been suggested. Here, we review 30 years of research on tocopherols in model and crop species, with emphasis on the improvement of vitamin E content using transgenic approaches and classical breeding. We will discuss future prospects to further improve the nutritional value of our food.Tocopherols, together with tocotrienols and plastochromanols belong to a group of lipophilic compounds also called tocochromanols or vitamin E. Considered to be one of the most powerful antioxidants, tocochromanols are solely synthesized by photosynthetic organisms including plants, algae, and cyanobacteria and, therefore, are an essential component in the human diet. Tocochromanols potent antioxidative properties are due to their ability to interact with polyunsaturated acyl groups and scavenge lipid peroxyl radicals and quench reactive oxygen species (ROS), thus protecting fatty acids from lipid peroxidation. In the plant model species  Tocochromanols with a fully saturated side chain are called tocopherols and those with an unsaturated side chain tocotrienols. The number of methyl groups in the chromanol ring defines the four natural occurring α, β, γ, and δ-tocopherol and tocotrienol subforms [Four tocopherols and four tocotrienols form a group of lipid-soluble antioxidants called tocochromanols, commonly known as vitamin E ,2. Theirsubforms . Vitaminsubforms . It is gsubforms .In this review, we follow the elucidation of the tocopherol biosynthesis pathway genes over the last 30 years of research, with a particular focus on their function and their involvement in plant processes. We will focus on the biosynthesis bottlenecks detected and the transgenic and non-transgenic breeding efforts that have been undertaken in order to improve the antioxidant and nutritional values of important crops.p-hydroxyphenylpyruvate and is derived from tyrosine degradation [Arabidopsis thaliana , this step is catalyzed sequentially by two phytol kinases [Tocopherol biosynthesis is initiated in the plant’s cytoplasm, but, except for this first step, its biosynthesis takes place in the plastids. Here, the necessary enzymes are localized at the inner envelope or in the plastoglobules ,8,9. Theradation ,11. The radation ,13. In A kinases . The two1O2), which oxidizes, amongst others, membrane lipids, proteins, amino acids, nucleic acids, nucleotides, and carbohydrates [1O2 results in the corresponding tocopherol quinones (and other derivatives), some of which have been shown to be potent antioxidants [Tocopherols are one of the most valuable antioxidants because of their remarkable chemical mode of action. They interact with polyunsaturated acyl groups and protect polyunsaturated fatty acids from lipid peroxidation by scavenging lipid peroxy radicals and quenching ROS produced e.g., by photosystem II and during membrane lipid peroxidation ,35. Durihydrates ,40. The oxidants ,42,43.Euphorbia, or grape, α-tocopherol is the main tocopherol form in seeds [Tocopherols occur in mammalians, photosynthetic bacteria, fungi, algae, and plants, but only photosynthetic organisms are able to synthesize them ,3. The cin seeds ,47,48,49in seeds ,32,50,51in seeds ,53,54,55Since 1922, when the term vitamin E was introduced by Evans and Bishop , the rolAccording to the German Society for Nutrition (Deutsche Gesellschaft für Ernährung e.V.), the recommended daily amount of vitamin E for an adult between 25 and 51 years is 14 mg/day. Nuts, seeds, and vegetable oils are among the best sources for the dietary uptake but also green leafy vegetables, fruits, and cereals. Soybean, rapeseed, and corn oil are the best dietary sources for γ-tocopherol, whereas the most biologically active form of vitamin E, α-tocopherol, can be found in wheat germ oil, sunflower, and olive oils ,73. Of tIn leaves, tocopherols are located in chloroplast membranes, plastoglobules, and thylakoid membranes and are thus in close proximity to the photosynthetic apparatus. For this reason, it was believed that tocopherols were the most important antioxidants in plants for preventing damage to the apparatus from photosynthesis-derived ROS. However, recent studies indicate that tocopherols only partially contribute to a considerable variety of plant antioxidants such as carotenoids, ascorbates, glutathiones, and flavonoids, which also contribute to photo-protection ,75,76. NStep 1: Homogentisic Acid SynthesisPDS1, known to encode the enzyme p-hydroxyphenylpyruvate dioxygenase (HPPD) and was initially detected in carrot cells [A. thaliana pds1 mutant was devoid of tocopherols and plastoquinones and had a lethal photobleached phenotype, thus showing that PDS1 is a key gene of the tocopherol biosynthesis pathway [Escherichia coli (E. coli) expression studies, the protein HPPD catalyzed the accumulation of HGA. The subsequent oxidative polymerization of HGA to an ochronotic pigment resulted in a brownish coloration of the growth medium [The first committed step of the tocopherol biosynthesis is initiated by the gene ot cells . The cor pathway ,17. In Eh medium ,80,81,82PDS1 in A. thaliana plants led to elevated tocopherol concentrations of up to 43% in leaves and 28% in seeds, without a change in tocopherol composition [A. thaliana seeds resulted only in slightly increased tocopherol concentrations [PDS1 was co-expressed with downstream genes within the tocopherol biosynthetic pathway, the tocopherol content in seeds of A. thaliana, rapeseed, and corn kernels increased up to 1.8-, 2.0- and 3-fold, respectively [Constitutive expression of position . In othetrations ,84,85. Itrations . Higher ectively ,87,88.PDS1 is also linked to fruit ripening as well as leaf senescence in rice and barley [PDS1, indicating that these compounds can affect tocopherol biosynthesis.The expression of d barley ,81,89. Bd barley ,81. ThisStep 2: Polyprenyl Side Chain SynthesisA. thaliana seedlings with labeled PDP demonstrated the integration of free phytol into tocopherols [A. thaliana vte5-1 mutant plant, which had a 20% reduced seed tocopherol concentration compared to the wild type and increased amounts of free phytol. The corresponding VTE5 protein had a high homology to a dolichol kinase and was able to catalyze the phosphorylation of free phytol into PDP [Uptake experiments of cotyledon-derived soybean suspension cultures with different tocopherol precursors demonstrated that PDP, together with HGA, has a high impact on tocopherol synthesis . PDP, thopherols . Furtherinto PDP .VTE5-like knockdown genes and are deficient in leaf and fruit tocopherol displayed multifaceted impacted metabolisms. While the chlorophyll content was not affected, the knockdown resulted in an alteration of the prenyl lipid profile in fruits and in an increase in the fatty acid phytyl ester synthesis in leaves, leading to changes in photosynthesis and sugar partitioning, which in turn affected tomato fruit quality [Interestingly, tomato plants that have  quality .vte6 mutants might be an effect of phytol accumulation rather than tocopherol shortage, resulting in a disruption of chlorophyll and galactolipid synthesis in chloroplast membranes. Recent studies also showed that tocopherol levels are indirectly regulated through the contribution of chlorophyll synthases by reducing PDP precursors, thus providing new possible alternative routes for improving tocopherol content [The long-missing second enzyme of the phosphorylation pathway, phytyl phosphate kinase (VTE6) has recently been discovered using a phylogenetic approach . The tra content ,95.Step 3: MPBQ  SynthesisVTE2 [A. thaliana plants lacking VTE2 are completely deficient in all tocopherol derivatives and all pathway precursors, indicating that this is a limiting step in tocopherol biosynthesis [VTE2 mRNA levels by antisense RNA led to a 10-fold reduction of tocopherol content in A. thaliana seeds. In contrast, overexpression of VTE2 resulted in up to a 2-fold increased tocopherol concentration in seeds and up to a 4.4-fold increase in leaves [VTE2 gene from the bacteria Synechocystis sp. PCC 6803, suggesting species-specific differences in tocopherol biosynthesis regulation [LsHPT) caused an increase up to 4-fold of the α- and 2.6-fold of γ-tocopherol content in lettuce leaves in comparison to the non-transformed plants [The condensation of HGA and PDP to MPBQ is catalyzed by the enzyme HPT, which is encoded by the gene VTE2 ,97. A. tynthesis . Transgen leaves ,22. Integulation . The cond plants .A. thaliana vte2 mutants made their functional role particularly apparent in those plants. Seedlings exhibited severe defects during germination and early seedling growth due to high concentrations of non-enzymatic lipid peroxides and hydroxy fatty acids, indicating that tocopherols are crucial for the prevention of lipid oxidation during these important developmental stages. The seedlings were disturbed in root growth and in cotyledon expansion and showed a slowed storage lipid catabolism [vte2 mutants compared to the wild type, probably due to the non-enzymatic oxidation of storage lipids. The accumulation of lipid peroxidation products in vte2 mutants was correlated with an increased expression of many defense-related genes, showing another main role of tocopherols in the regulation of plant defense responses by modulating the levels of lipid peroxidation products [vte2 are abolished if tri-unsaturated fatty acids are suppressed as shown in the quadruple mutant fad3-2fad7-2fad8vte2-1, proving that there is a vte2-specific phenotype [vte2 plants [The lack of tocopherols in tabolism . Furtherproducts . The devhenotype . The pre2 plants . PC-8 ha2 plants ,103.vte2 plants are indistinguishable from wild-type plants, confirming that tocopherols are substitutable by other antioxidants at this developmental stage [Under normal growth conditions, as well as high light stress, adult al stage ,27,32. Hal stage ,104. MaeStep 4: DMPBQ  SynthesisSynechocystis sp. PCC 6803 and shares substrate specificities with the corresponding protein in A. thaliana, MPBQ methyltransferase [VTE3 and without the catalysis of the prenylquinol DMPBQ, α- and γ-tocopherol could not be produced. The enzyme is also involved in plastoquinone synthesis and is able to use 2-methyl-6-solanesyl-1,4-benzoquinol (MSBQ) for the formation of plastoquinol-9 (PQ-9).The enzyme that methylates MPBQ to DMPBQ was first identified in nsferase ,26. The A. thaliana vte3 mutants were isolated that had different phenotypes according to their origin and the severity of the mutation. Plants with a mutated vet3-1 allele, derived from ethyl methanesulfonate (EMS) mutagenesis, were found to have a partial loss-of-function of the enzyme. As a result, an altered tocopherol composition along with reduced MPBQ/MSBQ methyltransferase activity was observed. In these plants, both δ- and β-tocopherol concentrations in leaves were increased at the expense of α-tocopherol, whereas γ-tocopherol was replaced by higher δ-tocopherol levels, but PQ amounts were not affected in seeds [vte3-2 allele or Ds-tagged A. thaliana plants, which have a complete gene disruption, were deficient in α- and γ-tocopherol, as well as PQ. These mutants exhibited a pale green phenotype, abnormal chloroplasts and did not survive beyond the seedling stage [Several in seeds ,26. In cng stage ,25.VTE3 in A. thaliana and soybean did not have a substantial impact on the tocopherol amounts but severely changed the tocopherol composition [A. thaliana VTE3 and VTE4 (γ-TMT) in soybean seeds shifted the tocopherol composition in favor of α-tocopherol (>90%). This is especially of interest for the genetic engineering of crop plants, in which seeds are naturally enriched in γ-tocopherol . The nutritional value of those plants could be enhanced by changing the composition towards α-tocopherol.The overexpression of position ,75. In sposition . The seeStep 5: Synthesis of γ-Tocopherol and δ-Tocopherolvte2 mutant plants, vte1 mutant plants lack all tocopherol forms due to the absence of a functional tocopherol cyclase (TC) enzyme. They accumulate the intermediate product DMPBQ, the un-cyclized precursor of γ-tocopherol, instead [AtVTE1 cDNA in E. coli efficiently converted DMPBQ into γ-tocopherol, demonstrating the cyclase activity of the enzyme [VTE1 gene from A. thaliana and maize (Zea mays) in transgenic rapeseed (Brassica napus) increased concentrations of all tocopherol forms in the seed oil of T1 and T2 generation plants. However, using different B. napus VTE1 alleles, only a 1.5-fold increase in seed α-tocopherol was produced [Similar to the  instead . The expe enzyme ,55,105. produced ,105.VTE1 gene in A. thaliana plants under a constitutive promoter resulted in a 7-fold increase of the leaf tocopherol concentration and an extreme shift from α- to γ-tocopherol [A. thaliana vte1 mutants, whereas PC-8 accumulates with tocopherols in VTE1-overexpressing plants, showing that TC is also able to cycle PQ-9 into PC-8 [AtVTE1 overexpression in rapeseed induces a 2.4-fold increase in PC-8 levels [vte1 mutants are indeed caused due to the absence of tocopherols [Interestingly, expression of the endogenous copherol . The reacopherol . PC-8 shnto PC-8 ,106. Acc8 levels . NeverthA. thaliana vte1 mutant is not inhibited in its development, despite being deficient in tocopherols and 1O2 accumulation in chloroplasts [vte1 mutants resulted in an increase of ascorbate and glutathione, which belong to the most abundant antioxidants in higher plants. This effect was reversed when VTE1 was overexpressed: their concentrations were reduced to 40–60% relative to wild-type plants [A. thaliana vte1 mutants caused increased lipid peroxidation in water-stressed plants [VTE1 led to enhanced tolerance against salt stress in rice and drought stress in tobacco plants, providing evidence that crop species with increased tolerance to environmental stresses as well as high tocopherol content could be developed [The roplasts . Germinaroplasts ,27,108. roplasts . Furthere plants ,75. It ie plants . Hence, d plants . Furthereveloped ,111.A. thaliana vte1 mutants revealed a different role of tocopherols and indicated that α-tocopherol may have an impact on cellular signaling by altering plant hormone levels [Intriguingly, another analysis of e levels ,113. In e levels . The role levels ,116. Thee levels .sucrose export defective1 (SXD1) led to the suggestion that tocopherols might play a role in regulating carbon translocation. The maize sxd1 mutant has a vte1 mutant resembling phenotype that is characterized, amongst others, by the leaf-specific accumulation of starch and anthocyanin, and the deposition of callose in phloem parenchyma transfer cells. These characteristics are coincident with a loss of the symplastic transport, as well as a typical increase of the soluble sugar content due to the sucrose export deficiency [StSXD1 expression in potato also induces defects in carbohydrate transport [The analysis of the maize gene ficiency ,118,119.ransport , showingransport .Step 6: Synthesis of α-Tocopherol and β-TocopherolVTE4 and encodes a key enzyme (γ-TMT) that methylates both δ- and γ-tocopherol to β- and α-tocopherol, respectively . This confirms that VTE4 expression in sunflower seeds directly regulates the amount of α-tocopherol.The exclusive overexpression of the  content . Seed-spthaliana ,33. Becathaliana . In thesVTE2 overexpressing plants but not when VTE2 and VTE4 were simultaneously expressed [VTE2 and VTE4 was additive in A. thaliana leaves and seeds. The total tocopherol content increased up to 12-fold, while, at the same time, all of the γ-tocopherol was converted to α-tocopherol [VTE4 is suggested to be a key gene for metabolic engineering of enhanced α-tocopherol levels in crop plants. Similarly, using a transgenic approach, Endrigkeit et al., showed that a B. napus VTE4 homolog was able to increase the α-tocopherol content 50-fold in transgenic Arabidopsis seeds, providing a promising candidate for the marker-assisted selection of α-tocopherol content in rapeseed [The fact that γ-TMT activity is limiting, becomes particularly clear when plants are exposed to abiotic stress. High levels of γ- and δ-tocopherol levels were observed in stressed leaves of xpressed . Moreovecopherol ,75. Thusrapeseed .Improving vitamin E content in staple crops has been a major aim of crop breeding due to its benefits for health and oil quality . VitaminVTE3 alleles and detected that their differential expression is associated with differences in methylation of a short-interspersed nuclear element (SINE) retrotransposon located in the promoter region that is responsible for the regulation of vitamin E content in tomatoes [Moreover, Quadrana et al., analyzed tomatoes . In anottomatoes . These oVTE4 in B. juncea plants [VTE4 [VTE1 gene, rice and tobacco plants with an increased tolerance against salt and drought stress, respectively, and a simultaneously enhanced tocopherol content were developed [The vitamin E biosynthetic pathway has been well elucidated in model species, and select genes have been transformed and overexpressed individually or collectively in various plants to improve vitamin E content and composition ,148,149.a plants . In anotts [VTE4 ,153. Inteveloped ,111. TheIn contrast to attempts to improve α-tocopherol content, it is relatively difficult to significantly increase the total vitamin E content in crops. The most successful attempts at increasing total vitamin E content have occurred in rapeseed and soybean, with a nearly 2.4-fold and 15-fold enhancement, respectively, of total vitamin E content after co-overexpression of several key genes for vitamin E biosynthesis ,87. CollPresent studies using model plant species demonstrate that vitamin E biosynthesis genes are well understood. The use of mutant plants has helped with the discovery and analysis of the roles of tocopherols in plant processes beyond their antioxidant function. However, open questions remain about the specific functions of the different tocopherol forms as well as their concrete roles in signaling and defense responses. Additionally, plastoquinones and tocotrienols, which are known to be derived from the same head group as tocopherols, have gained attention in recent years but most of their roles still remain elusive ,154,155.Recently, new genes and epigenetic mechanisms have been discovered through investigations of the chlorophyll degradation pathway, transgenic and phylogenetic approaches, or by conducting joint linkage/genome-wide association studies ,91,95. MB. napus, and can now be used for marker-assisted breeding [Natural variations of vitamin E content have been investigated and demonstrated wide variations in/among different crops, which suggest a tremendous genetic potential for breeding vitamin E improved crops. On this basis, several QTL/association mapping studies have been carried out and have detected a range of genetic loci associated with vitamin E content and composition, but of which many detected loci cannot pinpoint candidate genes ,158,159.breeding ,126.To date, many crops have been sequenced including vitamin E-rich staple crops, such as rapeseed, soybean, maize, and rice ,162,163.


The study aimed to monitor circulating tumor cells (CTCs) in early stage lung adenocarcinoma patients.P < 0.01). Additionally, CTCs detected by CanPatrolTM could be validated by CytoploRare or Pep@MNPs.CTCs were characterized and classified to epithelial (E-) CTCs, mesenchymal (M-) CTCs and epithelial- mesenchymal (E&M-) CTCs, as per epithelial-mesenchymal transition(EMT) biomarkers. CTCs could not be found in healthy controls. However, in cohort A, CTCs were found in 17 (17/18) cases. Detection rate of E-CTCs was lower (5/18) compared with M-CTC (10/18) or E&M-CTC (14/18). Highly abundant M-CTCs were prone to being in the tumors > 2 cm. In cohorts A and B, CTCs count increased significantly in all patients with tumor progression (7/7). Higher CTCs level or change range could be found postoperatively in the patients with tumor progression, as compared with patients with disease free survival -CTCs by using CytoploRare and EPCAM(+)-CTCs by using Pep@MNPs prior to operation, in cohorts C and D, respectively.We included four cohorts of patients and 20 healthy controls. In cohort A, CTCs were detected by a newly established approach, i.e., CanPatrolin situ, and CTCs detection can effectively monitor tumor progression. The distinguishing of biomarkers of highly invasive and aggressive CTCs warrants further robust study.CTCs can be detected in early stage lung adenocarcinoma, even in adenocarcinoma  In 2015–2016, 224,390 cases were newly diagnosed with lung cancer in USA [7 white blood cells per milliliter of blood [® and Adnatest®,and EpCAM-independent detection methods, e.g., ISET® and ScreenCell®. However, the sensitivities of EpCAM-based detection methods seemed to be significantly lower than EpCAM-independent detection methods [Circulating tumor cells (CTCs), which shed from the primary tumor into the vasculature or lymphatics, can be regarded as a new prognostic factors of metastatic process . CTCs weof blood –7, hence methods –11 due tTM  [in situ (AIS) of lung. Additionally, a longitudinal study was performed to assess the clinical implications of continuous monitoring of CTCs. Furthermore, we validated CanPatrolTM by using other two ways to detect Folate receptor (FOLR) (+)-CTCs [In stage 1A and 1B lung cancer, the detection of CTCs has found to be a sensitive biomarker to predict the prognosis , 13. In , China) , 15, and(+)-CTCs , 17 and (+)-CTCs , respectin situ hybridization clearly identified EMT markers in an epithelial (E-) CTC (red dots), a mesenchymal (M-) CTC (green dots) and an epithelial- mesenchymal (E&M-) CTC(red and green dots), respectively. Therefore, these CTCs were classified as E-CTCs, M-CTCs and E&M-CTCs, respectively. In cohort A, E-CTCs, M-CTCs and E&M-CTCs were detected in 5,10 and 14 cases, respectively. There were no statistically significant correlations between the number of CTCs and tumor size, age and gender.CTCs could not be found in 20 healthy controls, but were detectable in 17(17/18) cases in cohort A, as shown in Table Figure As shown in Figure We performed longitudinal studies in 14 cases of cohort A after operation as shown in Figure However, as compared with before operation, cases A5, A11 and A12 had significant increase of CTCs after operation Figure , while tCases A3 and A8 respectively had a recurrence in six months and one year after the operation, proved by the increased CTCs Figure , computeLongitudinal CTCs monitoring was performed in case B1 as shown in Figure In cases B2-B5, CTCs count significantly increased after operation Figure . IntriguP < 0.01). Furthermore, change range of CTCs in patients with tumor progression was significantly higher as compared to patients with disease free survival .In cases B6-B19 with disease free survival, CTCs count significantly decreased in cases B10-B12, slightly increased in cases B8, B9, B13, B15, B17, B18 and B19, or remained stable in cases B6, B7, B14, and B16, as shown in Figure TM, CytoploRare and Pep@MNPs, as EMT-CTCs, (FOLR)(+)-CTCs and EPCAM(+)-CTCs, respectively. As shown in Figure TM were not detectable but EpCAM(+)-CTCs were positive, suggesting Pep@MNPs probably is more capable of capturing epithelial CTCs. Totally, our results demonstrated CTCs detected by CanPatrolTM can be validated by CytoploRare or Pep@MNPs in lung cancer patients.For distinguishing, we entitled the detected CTCs by CanPatrolin situ, melanoma in situ and bladder cancer in situ , vimentin and twist. EpCAM and CKs are commonly expressed in CTCs from epithelial-derived malignancies [® was especially lower, compared with ISET® as shown in Online Table TM can be validated by CytoploRare or Pep@MNPs in lung cancer patients. However, the sensitivity and specificity of CTCs detected by these methods warrant further study.Currently, technologies for CTCs detection can be divided into EpCAM-based and EpCAM-independent methods, respectively. However, the sensitivity of EpCAM-based detection methods seemed to be relatively low ranging from 21% to 41% , 9, showgnancies , 15. Vimgnancies , 15. Twignancies . In the ® system to detect CTCs before and after surgery, and found no statistical correlation between EpCAM (+) CTCs count and survival or recurrence, demonstrating EpCAM (−) CTCs or M-CTCs also should be evaluated to monitor tumor progression. In our longitudinal study, CTCs decreased significantly after operation, probably due to the reduced tumor burden, and CTCs significantly increased in all the cases (6/6) of cohorts A and B with tumor relapse or progression, demonstrating CTCs detection can effectively monitor tumor activity.After surgical removal of tumor mass, some early-stage NSCLC patients will relapse or develop metastases finally. It emphasizes the importance of risk assessment of tumor progression in these patients, and CTCs are considered as the predictive biomarker for guiding cancer treatment strategies . CTCs coin vitro [Theoretically and empirically, CTCs can be erased by anoikis, i.e., a form of programmed cell death that occurs in anchorage-dependent cells when they detach from the surrounding extracellular matrix . As showin vitro . WhetherTM can be validated by CytoploRare or Pep@MNPs in lung cancer patients. The distinguishing of biomarkers of highly invasive and aggressive CTCs warrants further robust study.Collectively, CTCs could not be found in 20 healthy controls, but were preoperatively found in 94% (17/18) cases with lung adenocarcinoma in early stage. E-CTCs, M-CTCs and E&M-CTCs were detected in 27.8% (5/18), 55.5% (10/18) and 77.8 (14/18) cases, respectively. Highly abundant M-CTCs were prone to being in the tumor ≥ 2 cm, compared with tumors < 2 cm, suggesting T1b lung adenocarcinoma may be prone to shedding of highly invasive and aggressive CTCs. Additionally, CTCs detection could effectively monitor tumor progression. Our results demonstrated CanPatrolFrom January 2013 to August 2016, four cohorts of patients  and 20 healthy controls (non-smokers) were enrolled, respectively as shown in Table Cohorts A and B included 18 and 19 patients with stage I lung adenocarcinoma, respectively (+)-CTCs and EPCAM(+)-CTCs, respectively. In cohorts C and D, we simultaneously detected EMT-CTCs&FOLR(+)-CTCs and EMT-CTCs&EPCAM(+)-CTCs prior to operation, respectively. In addition, EMT-CTCs had been detected in the healthy controls.To distinguish the detected CTCs by using CanPatrolAll the cases, except Case A10 who had CT-guided percutaneous lung biopsy prior to operation, did not undergo invasive diagnostic procedure. All the cases underwent video-assisted thoracoscopic surgery(VATS). i.e., lobectomy and lymphadenectomy. The diagnosis was confirmed pathologically in all the cases. The clinical and demographical characteristics were shown in Table Ten ml of blood was collected from the healthy controls and cases, and transferred into sample preservative tubes  containing ammonium chloride-based lysing buffer by a tailored connection device  and incubated at room temperature for 30 min.TM was used to detect EMT-CTCs, which is a newly established technology to detect CTCs, containing the following steps [in situ hybridization (ISH), based on the branched DNA (bDNA) signal amplification technology, to detect EMT markers, e.g., cytokeratins(CK) 8, 18 and 19, epithelial cell adhesion molecule (EpCAM), vimentin and twist.CanPatrol ng steps , 15: (1)TM was depicted in the recently published protocol [The details of classification of EMT-CTCs by using CanPatrol protocol . FinallyFOLR(+)-CTCs were detected by using CytoploRare method provided by GenoSaber Biotech Co. Ltd.  , 17. BloFirstly, erythrocytes were removed by red blood cell lysis and CD45+ leukocytes were depleted by using a magnetic bead separation method. Then it was labeled with a conjugate of a tumor-specific ligand folic acid and a synthesized oligonucleotide , 17. TheIn this study, we used an arbitrarily defined CTC unit, which was defined as the number of CTCs detected in 3 ml of blood. FOLR value > 8.7 FU/3 ml was defined as positive , 17.EpCAM(+)–CTCs were isolated by using the EpCAM recognition peptide functionalized iron oxide magnetic nanoparticles (MNPs) (Pep@MNPs) . 10 μLof


Deletions in the 4p16.3 region are associated with Wolf-Hirschhorn syndrome (WHS), a contiguous gene deletion syndrome involving variable size deletions. In this study, we perform a cytogenomic integrative analysis combining classical cytogenetic methods, fluorescence in situ hybridization (FISH), chromosomal microarray analysis (CMA), and systems biology strategies, to establish the cytogenomic profile involving the 4p16.3 critical region and suggest WHS-related intracellular cell signaling cascades. The cytogenetic and clinical patient profiles were evaluated. We characterized 12 terminal deletions, one interstitial deletion, two ring chromosomes, and one classical translocation 4;8. CMA allowed delineation of the deletions, which ranged from 3.7 to 25.6 Mb with breakpoints from 4p16.3 to 4p15.33. Furthermore, the smallest region of overlapping (SRO) encompassed seven genes in a terminal region of 330 kb in the 4p16.3 region, suggesting a region of susceptibility to convulsions and microcephaly. Therefore, molecular interaction networks and topological analysis were performed to understand these WHS-related symptoms. Our results suggest that specific cell signaling pathways including dopamine receptor, NAD+ nucleosidase activity, and fibroblast growth factor-activated receptor activity are associated with the diverse pathological WHS phenotypes and their symptoms. Additionally, we identified 29 hub-bottlenecks (H-B) nodes with a major role in WHS. Wolf-Hirschhorn syndrome  is a well known genetic condition with estimated prevalence of 1/20,000 to 1/50,000 births  and Wolf (NELFA). WHSC1 is involved in controlling the expression of several genes and acts as an agent in chromatin remodeling  levels, dysfunctional mitochondrial transition-pore opening, and hyperpolarization [ LETM1 without seizures, or patients with seizures and the presence of LETM1 have been described [ (PIGG), complexin 1 (CPLX1), and LETM1 genes, frequently associated with WHS convulsions [In human cell lines, the deletion ofrization , 32. Furrization , 32. Allescribed , 33, 34.vulsions . WHSC1 and NELFA, in this region has already been associated with this condition [ WHSC1 shows transcriptional corepressor activity by expressing a histone methyltransferase [ NELFA encodes a member of the negative elongation factor involved in regulating the progression of transcription by RNA polymerase II [ NELFA in cell lines from patients with WHS is associated with delayed progression from the S- into the M-phase and altered chromatin assembly [Furthermore, our results showed a 330 kb region also associated with microcephaly. The deletion of two genes,ondition , 19, 35.nsferase , 36, whinsferase . NELFA eerase II  and histerase II . The hap (FGFR3), N-acetyltransferase 8 like (NAT8L), DNA polymerase Nu (POLN), and chromosome 4 open reading frame 48 (C4ORF48I). However, these genes are not directly associated with seizures and microcephaly but have a putative involvement in the skeletal development and plasticity of the human brain [The following four additional genes are also located in the susceptibility region proposed in this study, fibroblast growth factor receptor 3an brain –40. In aan brain .p value corrected = 0.002); (2) NAD(P)+ nucleosidase activity . These pathways participate in nicotinamide metabolism and calcium signaling, thereby contributing to excitability, exocytosis, motility, apoptosis, and cell transcription mechanisms [p value corrected = 0.006) and (4) FGF binding , which is involved in the maintenance of tissue homeostasis and regulation of metabolic processes with specific roles such as the regulation of cell migration, proliferation, and differentiation [Thus, focusing on the critical 4p16.3 chromosome region mapped in our study, we explored the molecular interaction networks and biological pathways involving WHS-associated genes. An initial list of 343 genes obtained from the GENCODE V24-GRCh38/hg38-UCSC database was used to construct an interactome network. This network was composed of 136 nodes and 750 edges . It is ichanisms , 43; (3)ntiation , 45. FGFR3 and FGFRL1, which interact with FGFs to activate a downstream signaling cascade. The hemizygous deletions of FGFR3 and FGFRL1 are implicated in the skeletal abnormalities and facial features typical of WHS [p value corrected = 0.001) has been implicated in numerous neurological processes including sleep regulation, feeding, attention, cognitive functions, olfaction, and hormonal regulation [p value corrected = 0.001) and molecular functions such as nuclear DNA replication  encompassing the stem-loop binding protein (SLBP) and NELFA genes, often deleted in WHS, were also predicted. It was not possible to predict GO and pathways categories for cluster 2.The receptors involved in these pathways are encoded byl of WHS , 46–48. gulation . Toxin t NELFA gene was localized in the SRO defined in this study, and the SLBP gene was identified as an H-B in the centrality analysis , which can be reduced by siRNA-mediated knockdown of the NELF-E component of the NELF complex , 51. Thiub WHSC1  using ceub WHSC1  [1, 17, The centrality analysis revealed that 90% and 10% of the H-B are located on chromosome 4 and other chromosomes, respectively. Considering this evidence, we suggest that the diversity of the pathological WHS phenotypes could be dependent on interrelated and close H-Bs located on the same chromosome. To the best of our knowledge, this is the first report of a cytogenomic integrative network analysis using systems biology to study the critical region associated with WHS. Our study described the putative cell signaling pathways altered in WHS that contribute to the integrative understanding of the role of contiguous genes in the spectrum of this syndrome.LETM1, FGFR3, WHSC1, NELFA, C4orf48, NAT8LI, and POLN). The network topological analysis identified 29 H-Bs, and the candidate gene NELFA was included among these H-Bs. In addition, significant GO categories showed that several cell signaling pathways are responsible for the seizures and microcephaly in WHS.This study combined clinical data and integrative analysis with conventional cytogenetic techniques, CMA, and systems biology strategies. We confined the region of susceptibility for microcephaly and seizures to a 330 kb sized region that encompassed seven candidate genes (


Congenital adrenal hyperplasia (CAH) is classified as classical CAH and non-classical CAH (NCCAH). In the classical type, the most severe form comprises both salt-wasting and simple virilizing forms. In the non-classical form, diagnosis can be more confusing because the patient may remain asymptomatic or the condition may be associated with signs of androgen excess in the postnatal period or in the later stages of life. This review paper will include information on clinical findings, symptoms, diagnostic approaches, and treatment modules of NCCAH. Congenital adrenal hyperplasia (CAH) is a group of diseases which develop as a result of deficiency of enzymes or cofactor proteins required for cortisol biosynthesis ,2,3,4,5.Non-classical CAH (NCCAH) includes a series of diseases occurring due to gene mutations or disorders in the steroid synthesis steps of steroidogenic acute regulatory protein (StAR) providing the transfer of cholesterol from mitochondrial membrane to the cell. Although NCCAH occurs in the deficiencies of 21-OHD, 11β-hydroxysteroid dehydrogenase (11β-HSD) and 3-beta hydroxysteroid dehydrogenase (3β-HSD), and StAR mutations, it is most commonly observed in 21 and 11-b OHSD deficiencies. Its prevalence is reported as 1/1000 . HoweverFindings of genital virilization are not observed at birth in NCCAH patients. Although premature pubarche was detected in a 6-month-old infant as the earliest example, clinical findings and symptoms in NCCAH cases usually start from the age of 5 and usually emerge in late childhood, adolescence, and adulthood ,9. Resul21-hydroxylase gene mutation was detected in 8.3% of the girls with premature pubarche  with premature pubarche, hirsutism, or polycystic ovary syndrome (PCOS) presentation . Tallness, accelerated bone maturation, development of premature pubic and axillary hair, and adult apocrine odor are findings which may be noted in both girls and boys in the prepubertal period. Heterozygote non-classical pubarche . CYP21A2entation . Increasentation  which anPubertal girls affected with NCCAH typically present with hirsutism ,25. It iIt may be difficult to distinguish the clinical symptoms and findings of premature adrenarche from those of PCOS in girls . Althougrd and 5th postmenstruation days)  on an empty stomach and at the follicular phase in menstruating females (between the 3on days) . This ison days) . Boys suon days) . A basalon days) ,33,34. Ion days) . The conon days) . NCCAH won days) . After e studies . Howeverriterion . ACTH anpa et al  reportedth minute 11-deoxycortisol level is higher than 18 ng/mL in the ACTH test, the diagnosis becomes definitive , immunochemiluminometric assay (ICMA), electrochemiluminescence immunoassay (ECLIA)] methods. Interference is a common problem in RIA methodology, so there is a need for improvement in purification and extraction methodology. Recently, more reliable and accurate results were reported with liquid chromatography coupled with mass spectrometry (LC-MS/MS) devices ,43. AmbrCYP21A2–CYP21A1P mutations are generally investigated. Null 12 G splice, 1172N, P3OL, V28IL, P453S.Int2 mutations are observed most commonly and they are positive in 73-87% of the cases (CYP11b1) and 3-b hydroxysteroid dehydrogenase (HSD3b2) and StAR mutations observed more rarely should be studied  known as NCCAH can be considered to be cases of familial glucocorticoid deficiency . Also, iIt is known that NCCAH cases usually cannot be detected during CAH screenings made in the neonatal period. Held et al  have repth minute of the ACTH test does not exceed 18 mg/dL  and GnRH agonist treatment for their additive effect on final height. In a study where GH and GH+GnRH agonists were given to prepubertal 3 cases and pubertal 3 cases, improvement in predictive heights were observed when compared to an untreated group . However2/day, divided into 3 doses , decrease in bone density, fractures, obesity, dyslipidemia, insulin resistance, diabetes, increase in the thickness of intima media, hypertension, cardiovascular problems, early mortality risk, and tumorigenesis risk may arise ,59. AlthThe overall rate of miscarriages in NCCAH patients is 20%. NCCAH also increases the risk of classical and non-classical CAH for the offspring babies of affected mothers . For the


Our findings demonstrate the endemic diversity of West African WNV strains and support future investigations into (i) the nature of WNV emergence, (ii) neurological tropism, and (iii) host adaptation.The West Nile virus (WNV), isolated in 1937, is an arbovirus (arthropod-borne virus) that infects thousands of people each year. Despite its burden on global health, little is known about the virus’ biological and evolutionary dynamics. As several lineages are endemic in West Africa, we obtained the complete polyprotein sequence from three isolates from the early 1990s, each representing a different lineage. We then investigated differences in growth behavior and pathogenicity for four distinct West African lineages in arthropod (Ap61) and primate (Vero) cell lines, and in mice. We found that genetic differences, as well as viral-host interactions, could play a role in the biological properties in different WNV isolates  in vitro and in vivo results with relevant epidemiological and molecular data. Our findings highlight the existence of West African lineages with higher virulence and replicative efficiency in vitro and in vivo compared to lineages similar to circulating strains in the United States and Europe. Our investigation of four West African lineages of West Nile virus will help us better understand the biology of the virus and assess future epidemiological threats.The West Nile virus (WNV) can cause severe neurological diseases including meningitis, encephalitis, and acute flaccid paralysis. Differences in WNV genetics could play a role in the frequency of neurological symptoms from an infection. For the first time, we observed how geographically similar but genetically distinct lineages grow in cellular environments that agree with the transmission chain of West Nile virus—vertebrate-arthropod-vertebrate. We were able to connect our  Flavivirus of the family Flaviviridae. The WNV is a single-stranded, positive-sense RNA virus. The genomic RNA is about 11 kilobases (kb), containing one long open reading frame (ORF) flanked by 2 non-coding regions. This ORF encodes for a polyprotein, which is processed into three individual structural , and seven non-structural  proteins [West Nile virus (WNV) is a member of the Japanese Encephalitis virus (JEV) serocomplex and is a part of the genus West Nile fever disease (WN fever) is caused by the WNV. WN fever in humans can range from asymptomatic infections or mild acute febrile illness, to neurological diseases including meningitis, encephalitis, and acute flaccid paralysis –7. WNV’sWNV was first discovered and isolated from the blood of a woman suffering from febrile illness in 1937 in Uganda . Cases ost century, emergences of WN fever and encephalitis have been reported in Europe [Meanwhile, WNV continued to spread and cause WN disease and encephalitis in Europe, Asia, and Oceania . In the n Europe , with a n Europe , and sevn Europe –24, Hungn Europe  and Serbn Europe .WNV is biologically diverse; up to nine lineages have been proposed –30. HoweLineage 2 was exclusively reported in Africa up until 2004, until it was isolated from humans and bird populations in Hungary, Greece, and Italy ,23,33. Lth lineage was isolated from India, and is often identified as a distinct clade of lineage 1 (clade 1c) [th lineage, based on a small gene fragment, has been described from Spain [Besides lineages 1 and 2, there are lineages that are less widespread. Lineage 3, also referred to as Rabensburg virus, was repeatedly isolated in the Czech Republic –38. Linelade 1c) . A putatom Spain ,41.Culex perfuscus in Kedougou, Senegal in 1992 [th lineage, or sublineage of lineage 4, was isolated from Uranotaenia unguiculata mosquitoes in Austria [Koutango virus (lineage 7) was initially classified as a different virus, but is now a distinct lineage of WN virus ,42. Line Austria .th lineage circulating in Africa [Despite the presence of lineages 1, 2, 7 (Koutango) and a putative 8n Africa ,46,47, Wn Africa –50, withn Africa ,52. For n Africa –57.i.e. intrinsic genetic variations among lineages—host adaptation, movement of host populations, climate and ecological factors could play a role in viral replication, virulence, and the outcome of infection. The N-linked glycosylation site of the envelope protein may be associated with differences observed in: (i) WNV neuroinvasiveness in mice, (ii) viral replication, and (iii) transmission of WNV in mosquitoes [A recent study on the vector competence of African WNV lineages demonstrated that local mosquito populations lack efficient transmission of WNV . Besidessquitoes ,58–60. Iin vitro and in vivo models. Ultimately, understanding the relationships among ecological and genetic differences will ameliorate our understanding of WNV emergence, epidemiology, and its maintenance in nature.To address these questions, we analyzed complete coding regions (polyproteins) of four different lineages circulating in Senegal and West Africa. Using additional WNV sequences from Genbank, we performed a phylogenetic analysis using the complete polyprotein sequences of the viruses and investigated sites for positive selection. We also analyzed the biological properties of these 4 WNV lineages using Culex mosquito species, while the lineage 7 strain was isolated from a tick species.In this study, three complete polyprotein genes from Senegal isolates were sequenced: ArD76986, ArD96655, and ArD94343 . These nAcknowledging previous works that have reconstructed the evolutionary history and those that have characterized novel isolates and lineages of WNV, we included seven additional complete ORF sequences to compare differences at the gene and protein level . Among rThe 1937 WNV isolate of strain B956  is of particular interest, as it is the oldest clinical isolate available with a complete ORF sequenced. Amino acid pairwise identity was 93.9% to the NY99 strain sequence and to the lineage 1a (SN) sequence, 89% to the lineage 7 strain sequence and 91.1% to the lineage 8 strain sequence. At the nucleotide level, the lineage 2 strain (UG) had a pairwise identity of 79.1% to the NY99 sequence, 79.3% to the lineage 1a strain (SN) sequence, 76.9% to the lineage 7 strain sequence and 77.7% to the lineage 8 strain sequence. Additionally, B956 contains a 12 base pair deletion at nucleotide position 1,331, corresponding to the WNV envelope glycosylation site.nd and 72nd codon sites of the pre-membrane protein (prM) have been shown to play a role in enhancing the virulence and particle secretion in WNV [We compared published sequences and published works to identify whether mutations that have been shown to influence WNV virulence and replication were present in the newly sequenced open-reading frames. For each strain, we discovered amino acid changes that were associated to a phenotypical change and many additional mutations with unknown consequences . For exan in WNV . At thisth positions of the envelope (Env) protein, which is considered a virulence factor [th codon position of the NS3 protein [th codon position , a major split that diverges lineages 1, 5 and 7 from lineages 2, 3, 4, 8, and 9. Both lineage 1 and 2 show multiple introductions into Europe and other New World countries. Additionally, we see that lineages 1, 2, 7, and 8 have been isolated in West Africa, yet only lineages 1 and 2 have emigrated.A total of 95 sequences, including 3 novel polyprotein sequences from Senegalese isolates , were usi.e., total number of particle release)  of the lysed cell fracture to measure genome replication , qRT-PCRrelease) , immunofrelease) , and plarelease) . Using AAedes pseudoscutellaris cells, growth dynamics were similar for all lineages,  while the lineage 2 strain had higher genome replication in Vero cells . Lineage 8 showed a lower significant replication profile in Vero cells . Furthermore, differences in growth at T0 further supports that WNV lineages could have a preference to a specific cellular environment. The rate of viral attachment, entry and replication initiation can all depend on the genetics of the infecting strain [We observed cell-specific growth differences among different WNV strains. For example, g strain .in vitro models . Lineage 8 strain showed significantly lower genome copy numbers in Vero supernatants .We estimated the total number of released particles at different times post infection by measuring the WNV RNA copy number in the cell supernatant. All tested lineages had comparable genome copy numbers in Ap61 supernatants . Howeverp-value ranging from 3.98x10-13 to 0.011), while lineage 8 strain had significantly lower levels of protein translation in Vero cells. Nevertheless, the translation rate in lineage 8 increased significantly from T124-146 in Ap61 cells. We also noticed a delay on translation detection in both cells, with no detectable protein production until T99 hours  . The lin99 hours  and T50 50 hours  respectip-value ranging from 2.22x10-16 to 0.028)   in the supernatant fractions. In Ap61 cells, we found a similar profile of infectious particles production for all lineages, with significant higher rates at 124 hpi and 146 hpi for the lineage 7 strain (o 0.028) . In Veroo 0.049) .p-value ranging from 5.49x10-16 to 0.0223)  –73. We e 0.0223) . There aTo determine the virulence of WNV strains , we chalp-values < 0.05). In fact, the lineage 7 strain induced the shortest survival time compared to the other strains and always resulted in 100% mortality in every experiment  at 100 and 1000 PFU doses . In factdN) to synonymous (dS) substitutions per codon site  using 95 sequences, which represent all investigated WNV lineages. Our investigation on selection regimens acting on all WNV complete ORF sequences—with the FUBAR method—revealed 3313 well supported (posterior probability ≥ 0.9 and Bayes Factor < 3.0) sites under purifying selection  under diversifying episodic selection  and African green monkey kidney cells (Vero) to reflect infection dynamics in two common classes of WNV hosts (insect vector and primate)  Figs  and 4. Tte) Figs . We founte) Figs , 4 and 5utionary  and indiutionary .th and 446th codons in polyprotein gene (154-156th positions of the Env protein) that encode the N-glycosylation motif (NYS). This site is present in many lineage 1 strains, some neuroinvasive lineage 2 strains [Culex cells [Aedes albopictuscells [th codon in polyprotein (249th in NS3) was under diversifying selection (ω = 33.1) 13% of the time and under purifying selection (ω = -0.91) 87% of the time  and cause acute infections, we found that the majority of WNV codon sites are undergoing purifying selection . Neverth strains ,77, and ex cells  and betttuscells . This N-tuscells . Second,the time  in our W120PEPE123 motif), we did discover the P122S substitution in the lineage 8 strain  could lead to a better understand the transmission dynamics and maintenance cycles of WNV in nature. The low virulence phenotype of the lineage 8 strain could also be a factor for its consideration as a potential vaccine candidate for West Nile fever.The Senegalese lineage 1 strain exhibited moderate virulence in mice  and causin vitro and in vivo results. For example, future studies in cell lines with interferon may help clarify the impact of the S653F NS5 mutation for the lineage 7 strain. Additionally, the flavivirus 5’ and 3’ untranslated regions (UTR) can affect replication and translation; certain mutations in these regions can cause complete viral attenuation [Further studies could complement our analysis, particularly, on other factors that could explain differences in WNV host and disease dynamics. Exploring variations in codon usage bias could also help explain biological differences , as distenuation –87. Unfoin vivo, the West Nile virus is a pathogen with the capability to cause severe epidemics anywhere in the globe. As complete genome sequences including the 5’ and 3’ UTR regions are currently being generated, this could lead to future studies focused on in vivo transmission and growth dynamics. As additional strains of WNV are characterized, monitoring the global diversity and distribution will aid in threat assessment and epidemiological modeling if future outbreaks are to occur.Taking everything into account, especially differences in sequences, growth dynamics and virulence Aedes pseudocutellaris) were grown in L15 (Leibovitz’s 15) medium  and 10% tryptose phosphate  and incubated at 28°C without CO2. Vero cells   were grown using the same medium without tryptose phosphate and CO2. Furthermore, PS  cells were grown in same conditions than Vero cells and have been used for plaque assay.Two cell lines have been used for virus cultivation and growth kinetics. Ap61 cells (Aedes pseudoscutellaris (Ap61) continuous cells lines for 4 days. The infection status was tested by immunofluorescence assay (IFA), real-time RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction) and plaque assay. The supernatant of infected cells were aliquoted, frozen at -80°C, and used as viral stocks for growth kinetics.The virus strains used in this study corresponding to lineages 1, 2, Koutango (lineage 7) and 8 were described in A total of 862 complete WNV polyprotein gene sequences with country and year of isolation data were available and initially downloaded from Genbank for this study. A large number of sequences were from the Americas and formed a monophyletic group of lineage 1a comprising 770 sequences. To reduce computer-processing requirements while maintaining the authenticity of our results, we removed all lineage 1a sequences except for a single representative sequence denoted “NY99” (accession number: AF196835). With the addition of 3 new sequences, a total of 95 sequences were aligned using Muscle v3.8.31  and manuLikelihood mapping analyses for estimation of data quality were performed using Tree-Puzzle  ,91. For http://beast.bio.ed.ac.uk/Tracer) to ensure convergence during MCMC by reaching effective sample sizes greater than 100.The Bayesian phylogenetic analysis was performed using Bayesian Inference (BI) using a general time-reversible with gamma-distributed rate variation and invariant sites model (GTR+Γ+I), as selected by Akaike's information criterion (AICc) in jModelTest 0.1 . The evohttps://github.com/caiofreire.To reduce the number of sequences from the original 862 downloaded from Genbank, a maximum likelihood tree was estimated using FastTree v2.1.7  after id5 Ap61 or Vero cells in a volume of 400 μl of appropriate medium and infected with 2.4x103 PFU (plaque-forming unit) of virus in 400 μl of medium, resulting in a multiplicity of infection (MOI) of 0.01. After an incubation time of 4 hours, the medium was removed and replaced with 2 ml of new medium to set a zero point for the growth curves (T0). The harvesting of one well occurred at 22, 28, 50, 75, 99, 124, and 146 h post infection. Each harvest was performed as follows. Supernatants were removed and frozen at -80°C in small aliquots. Cells were washed once with phosphate-buffered saline (PBS) and then removed in 500 μl PBS. A volume of 20 μl of cell suspension was dried on a glass slide for a subsequent immunofluorescence assay as previously described [To perform this study and make it comparable with other studies ,96, viraescribed  to measu8, 5x104, 7.5x106 and 1010 PFU/ml. For each lineage, 2.4x103 PFU were used for kinetics in mosquito and mammal cells. The ratio of particles per infectious unit in the initial viral stocks ranged from 8 to 600 [in vitro infection could be explained to differences in the viral strain and to the infection conditions i.e. very low MOIs (0.01), and distinct cell lines.RNA was extracted from cell suspensions and supernatants and copy numbers of genome were quantified by real time RT-PCR as previously described . Infecti8 to 600 . Our vir8 to 600  and mosqad libitum. Infected animals were monitored daily for first signs of encephalitis  and death throughout the 21 days after infection. All statistical inferences were calculated using the Wilcoxon rank sum test.Mice were produced in the Institut Pasteur de Dakar farm, located in Mbao, approximately 15 kilometers from Dakar, Senegal. After one week of acclimatization, five-to-six-week-old Swiss mice were challenged by intraperitoneal (IP) injection with 100, 1000 and 10000 PFU of WNV lineages diluted in phosphate buffer saline + 0.2% endotoxin-free serum albumin (BSA). For each lineage and dose, two independent experiments of infection were made. Each individual experiment had 4 to 8 mice. A group of mice inoculated in parallel with an equivalent volume of phosphate buffer saline + 0.2% endotoxin-free serum albumin (BSA) was maintained as a control. Mice were kept on clean bedding and given food and water dN) over synonymous substitutions per synonymous site (dS) per codon sites. Briefly, sites with ω>1 are assumed to be under positive (diversifying) selection, and sites where ω<1 are undergoing negative (purifying) selection. When ω = 0, the site is undergoing neutral selection. To estimate ω, we applied three maximum likelihood methods: single likelihood ancestor counting (SLAC), fixed-effects likelihood (FEL), and internal fixed-effects likelihood (IFEL). We also investigated the presence of transient (episodic) selective pressures, using the mixed-effects model of evolution (MEME) [p-value ≤0.10. For FUBAR, sites were identified as undergoing positive selection when there was a posterior probability ≥0.90. All estimations were implemented using HyPhy v2.11 [To evaluate selection patterns on the complete coding sequences, we estimated the ratio of substitution rates (ω) per non-synonymous site (n (MEME)  and fastn (MEME)  approachhy v2.11 .Extraction of viral RNA from supernatants was performed with the QIAamp viral RNA mini kit  according to manufacturer’s instructions. For cell fractions, prior to RNA extraction, cells were lysed by serial cycles of freeze/thaw. For the detection and quantification of viral RNA, a consensus WNV real-time RT-PCR assay and corresponding RNA standard were used as previously described . The reaOverlapping RT-PCRs were done to recover the complete genome. All primer sequences can be found in the The PCR fragments were obtained using AMV reverse transcription kit  for reverse transcription and Go-Taq PCR kit  for amplification. The RT conditions were set according to the manufacturer’s instructions, and the PCR conditions were as follows: 5 minutes at 95°C, 40 cycles of 1 minute at 95°C, 1 minute at 53°C, 1 to 4 minutes (according the size of the PCR product) at 72°C, and 10 minutes at 72°C. The PCR products were purified from the agarose gel using the Gel extraction kit (Qiagen) and sequenced by Cogenics .Infected cells at different time points were dissolved in PBS and dropped on a glass slide. After complete drying, cells were fixed for at least 20 min in cold acetone, dried again, and then stored at -20°C until staining. Staining was done with a WNV-polyclonal mouse immune ascit diluted in PBS and incubated for 30 minutes at 37°C. After washing three times with PBS, cells were incubated with the second antibody , diluted 1:40 and blue Evans 1/100 in PBS, for 30 minutes at 37°C in the dark. The cells were washed again three times with PBS, dried, and covered with 50% glycerol in PBS. After dehydration, examination was done by fluorescence microscopy.KJ831223, FJ159131, AY277251, FJ159130, FJ159129, AY765264, KY703856, DQ176636, HM147823, FJ425721, KT207791, KJ934710, KP780840, KP780839, KT359349, KC496016, KC407673, KF179639, KJ883346, KC496015, HQ537483, KF647251, KT207792, JN858070, KP109692, KF179640, KM203863, KP780838, KP780837, KM203861, KM203862, JN393308, EF429197, EF429198, HM147824, EF429199, KM052152, EF429200, GQ903680, HM147822, GQ851605, DQ256376, GQ851604, JX041632, GQ851602, KT934796, KT934801, JX123031, JX123030, KT934800, KT934802, KT934803, GQ851603, KT934797, KT934799, KT934798, JX041628, JX041629, JX041630, HM051416, KT163243, EU249803, KC601756, JX442279, JX041634, KU588135, JQ928175, JN858069, KF647253, KC954092, JQ928174, JX556213, JF707789, FJ766331, FJ766332, JF719069, FJ483549, FJ483548, JF719066, JF719067, KF234080, GU011992, JF719068, DQ786573, AY701413, HM152775, AY701412, GQ851606, GQ851607, GQ379161, AF196835, KY703855, EU082200, KY703854, AY532665.S1 FigThe Shimodaira-Hasegawa values greater than 70% are shown at respective nodes. Tip labels are colored by proposed lineage. Sequences from (TIF)Click here for additional data file.S2 FigThe dashed line marks neutral selection (dN-dS = 0), points above the line (dN>dS) are under diversifying selection and below (dN<dS) are under purifying selection. The intensity of the point color is proportional to the posterior probability to observe that codon under the selection regimen, calculated with Fubar method.(TIF)Click here for additional data file.S3 FigUsing 95 WNV sequences, A) diversifying selection (dN>dS) and B) purifying selection (dN<dS) were estimated. The intensity of the point color is proportional to the posterior probability to observe that codon under the selection regimen, calculated with MEME method. Significant positively selected sites detected by other methods were also included in A).(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(CSV)Click here for additional data file.S3 TableThe NS5, envelope and NS5-partial 3’UTR regions were first amplified using flavivirus consensus or West Nile specific primers. This was followed by amplification of NS3 region using designed WNV primers. Finally, specific primers were designed according to the first sequences obtained and a second step of RT-PCR was done to obtain the complete genome.(DOC)Click here for additional data file.S1 DatasetRaw data for (XLSX)Click here for additional data file.S2 DatasetRaw data for (XLSX)Click here for additional data file.


The nuclear RNA exosome is an essential and versatile machinery that regulates maturation and degradation of a huge plethora of RNA species. The past two decades have witnessed remarkable progress in understanding the whole picture of its RNA substrates and the structural basis of its functions. In addition to the exosome itself, recent studies focusing on associated co-factors have been elucidating how the exosome is directed towards specific substrates. Moreover, it has been gradually realized that loss-of-function of exosome subunits affect multiple biological processes, such as the DNA damage response, R-loop resolution, maintenance of genome integrity, RNA export, translation, and cell differentiation. In this review, we summarize the current knowledge of the mechanisms of nuclear exosome-mediated RNA metabolism and discuss their physiological significance. Regulation of RNA maturation and degradation is a crucial step in gene expression. The nuclear RNA exosome has a central role in monitoring nearly every type of transcript produced by RNA polymerase I, II, and III . The exosome guarantees fidelity of the mature 3′ ends of certain stable RNA species, such as ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), telomeric RNAs, small nuclear and nucleolar RNAs (snRNAs and snoRNAs), not only by catalyzing 3′ end trimming, but also by degrading transcripts containing an incomplete 3′ end ,2,3. BesIntriguingly, recent advances in RNA sequencing techniques have enabled detection of novel Pol II transcripts , which aStrikingly, recent studies have been gradually revealing that the exosome is involved in multiple important biological processes. Those include the DNA damage response (DDR), R-loop resolution, maintenance of genome integrity, RNA export, translation, and cell differentiation. In this work, we review and update our current understanding regarding structural insights into RNA degradation by the exosome and its associated co-factors. We also summarize how abrogation of the functions of the exosome impacts cellular processes in mammals.The eukaryotic nuclear RNA exosome is a 3′–5′ exonuclease complex, consisting of a 9-protein catalytically inactive core complex (EXO-9) and two catalytic subunits, Rrp6 , and Dis3 . EXO-9 forms a double-layered barrel-like structure that comprises six ribonuclease (RNase) pleckstrin homology (PH)-like proteins  and three S1/K homology (KH) “cap” proteins  ,26,27. BSaccharomyces cerevisiae have revealed that RNA substrates show preferences for a specific path to Dis3 [Three additional co-factors, Mtr4 ), Rrp47 , and Mpp6, are required for maximal activity of the nuclear exosome. Rrp47 interacts with Rrp6 to provide a binding platform for Mtr4, an essential DExH-box RNA helicase . Mpp6 bi to Dis3 ,47. Nota to Dis3 : by asseThe fact that the exosome targets a wide variety of transcripts raises an important question: how is the exosome specifically recruited to particular RNA substrates? Recent studies have identified a number of nuclear exosome–adaptor complexes, which help the exosome load onto selective RNAs ,3,49. ThS. cerevisiae, and now is the most well-characterized co-factor that assists exosome-mediated RNA degradation and processing in budding yeast nuclei. Soon after recognizing the importance of polyadenylation of hypomodified methyonyl initiator transfer RNA (tRNAiMet) by the non-canonical poly(A) polymerase Trf4 for exosome-dependent tRNA quality control [Schizosaccharomyces pombe and humans , respectively. S. pombe has the Nrd1 homolog Seb1 [The Trf4/5-Air1/2-Mtr4 polyadenylation complex (TRAMP) was first described in  control , the ful control ,51,52. L control . Air1/2  control ,55,56. T control , such as control ,58,59, r control ,61, sn/s control ,63, telo control , CUTs [1 control ,59,64, a control ,66,67, a control ,71,72,73 control . Nrd1 an control ,76, wher control ,78,79. I control ,83,84,85 control . Nrd1 in control ,88. The  control . The Nrd control . Notablyd humans . Both S.log Seb1 , which hlog Seb1 ,92. Howelog Seb1 ,93,94. Flog Seb1 , remain log Seb1 ,97. Alsolog Seb1 . Howevernpl3-1 mutant strain. ISW1 is required to retain export-defective poly(A)-tailed RNAs on chromatin and remove them by recruiting the exosome. This finding implies that ISW1/exosome participates in a messenger ribonucleoprotein (mRNP) nuclear export surveillance system.Several other exosome partners exist in budding yeast. Utp18 and Nop53, an early and late associating small subunit processome factor, respectively, were shown to interact with the exosome to regulate ribosomal RNA precursor (pre-rRNA) processing . Both prS. pombe has a complex similar to S. cerevisiae TRAMP, consisting of Mtr4, Air1 and the Trf4/5 family of poly(A) polymerase Cid14 [S. pombe homolog of mRNA export factor Yra1 or ALYREF, was shown to interact with TRAMP to silence centromeric transcripts [S. cerevisiae [The fission yeast se Cid14 . It funcse Cid14 ,103,104 se Cid14 ,106, snose Cid14 , and Argse Cid14 . The prenscripts . Notablyrevisiae ,110, andS. pombe has a second Mtr4 homologue protein named Mtl1 (Mtr4-like protein 1), which is independent of TRAMP. Mtl1 interacts with the zinc-finger protein Red1 and various other proteins to form a complex called Mtl1–Red1 core (MTREC) or nuclear RNA silencing (NURS) [g (NURS) ,112. MTRg (NURS) . In agreg (NURS) . MTREC fg (NURS) ,112,113.g (NURS) ,115,116.g (NURS) ,118,119.g (NURS) . Red5 ang (NURS) ,122 and g (NURS) , which ag (NURS)   polymerase PAPD5 , respectively. These three proteins form the TRAMP-like complex . FunctioHuman TRAMP-like interacts with several additional proteins. It has been shown that TRAMP-like-mediated pre-rRNA processing is modulated by the AAA-ATPase NVL2 ,132 and S. pombe Red1, and therefore, Mtr4–ZFC3H1 is the human MTREC. Although RNA substrates of Mtr4–ZFC3H1 and NEXT partly overlap, there are clear differences in their features; Mtr4–ZFC3H1 substrates are longer in RNA body size and have a long poly(A)-tail [In the nucleoplasm, at least two distinct exosome adaptors are present. One is Mtr4–ZFC3H1 or poly(A) tail exosome targeting complex (PAXT), which brings the exosome to various kinds of lncRNAs, including snoRNA host gene (SNHG) transcripts, eRNAs , uaRNAs (A)-tail ,49. The (A)-tail ,143,144.(A)-tail ,143,144,(A)-tail ,49. Yet,(A)-tail , U-rich (A)-tail ,141, and(A)-tail . CBCA an(A)-tail ,146, and(A)-tail ,146,147.S. pombe Nrl1, also interacts with Mtr4. However, in contrast to the S. pombe counterpart, it is unlikely that Mtr4/NRDE2 associates with the whole exosome, since analysis using size-exclusion chromatography-coupled mass spectrometry (MS) revealed that Mtr4/NRDE2 elutes around 440 kDa, which is smaller than the exosome/Mtr4 complex (>600 kDa) [A nucleoplasmic protein NRDE2, which is the homolog of 600 kDa) . In agre600 kDa) .Loss-of-function of the exosome due to mutation or depletion of its subunits and co-factors can cause alterations in various biological processes , and ultcyclin D1 (CCND1) PROMPTs upregulated in response to DNA damage by ionizing irradiation provide a binding platform for the RNA-binding protein FUS/TLS. FUS/TLS recruited to the CCND1 promoter through PROMPTs represses the histone acetyltransferase activity of CBP/p300, which results in decreased CCND1 transcription [The activity of the nuclear exosome is altered during the cellular DDR. The change is triggered by phosphorylation of the NEXT subunit RBM7 by the stress-related kinase p38 MAPK/MK2 ,153. Phocription . Howevercription . This miSETX mutations in ataxia oculomotor apraxia 2 (AOA2) patients. It is speculated that SETX recruits the exosome to R loops to promote degradation of the RNA unwound and released by SETX, and thus prevents possible rehybridization and the resultant DNA damage.R loops are three-stranded structures composed of the nascent RNA hybridized with DNA template and the resultant displaced single-stranded DNA (ssDNA). R-loop resolution is a critical step to maintain genome integrity, since the displaced ssDNA is vulnerable to DNA damage ,158,159.Exosc3 (Rrp40) or Exosc10 (Rrp6), which allows conditional ablation of the exosome by drug treatment, loss of the exosome results in enhanced R-loop formation and genomic instability, due to an increase of ncRNAs associated with TSS and superenhancers [Over the last decade, the concept has emerged that exosome-mediated R-loop prevention is a critical step in immunoglobulin class switch recombination (CSR) and somatic hypermutation (SHM) in B lymphocytes . To initnhancers ,175,176.nhancers . In addition to its role in NEXT loading to nascent transcript 5′ ends, the CBC is required to initiate nuclear RNA export by recruiting various proteins. The TREX mRNA export complex is recruited to the 5′ end of mRNAs through the export adaptor proteins ALYREF and THO associating with CBC ,179,180.Recently, Sinturel et al. reported intriguing findings that diurnal oscillations in liver mass and hepatocyte size are regulated by rhythmic changes in ribosome biogenesis, in which the nuclear exosome plays a role . In thisGRHL3 transcripts, which encode a transcription factor critical for epidermal differentiation [EXOSC1/2/5 genes, is expressed at a low level in induced pluripotent stem cell clones generated from aged tissue donors, and the decreased expression of RNA exosome subunits causes the accumulation of AU-rich element-containing RNAs, including glutathione peroxidase 2 (Gpx2). Overexpression of GPX2 increases the reduced form of glutathione, thus scavenging glutathione-mediated reactive oxygen species, which consequently blunts the DDR and reduces apoptosis. Similar defects were observed following knockdown (KD) of exosome subunits EXOSC2 and/or EXOSC8 in ESCs. Mtr4 is also important in cell proliferation and differentiation. On the one hand, Mtr4 expression is highly upregulated when the self-renewal state of ESCs is induced by inhibitors of kinases, known as 2 inhibitors (2i) [Precise regulation of the activity and maintenance of the fidelity of gene expression is vital for stem cell self-renewal, differentiation, and development. Studies have suggested that the nuclear RNA exosome is essential for maintaining progenitor cell function and preventing premature differentiation. A defective exosome pathway can lead to aberrant accumulation of RNAs, among which are mRNAs encoding differentiation-specific transcription factors, and ultimately break the balance between proliferation and differentiation. For example, the nuclear exosome directly degrades ntiation . Depletintiation . They foors (2i) . On the ors (2i) . Additioors (2i) . All theors (2i) . These oEXOSC3 (Rrp40) mutation. Importantly, viral growth was suppressed in these cells, indicating the essential role of the exosome in viral biogenesis. NEXT-assisted exosome seems to be co-opted by the viral RNA polymerase, since similar results were obtained following RBM7 KD. Moreover, synthesis of host/viral chimeric transcripts generated as a result of “cap snatching”, in which initiation of viral transcription is primed using 5′ ends of host transcripts (cap with 10–20 downstream nucleotides), is decreased upon exosome-depletion. Collectively, these results suggest that the nuclear exosome coordinates with viral polymerase during the initial steps of viral transcription with Pol II at host promoters to enhance influenza A virus ribogenesis and infectivity. From the evolutionary point of view, viruses need to integrate their biological activities into hosts by recycling regulatory RNAs generated by hosts. The exosome, as the hub of RNA surveillance system, can be co-opted by viruses to facilitate the efficient formation of cellular/viral hybrid RNAs and cap-snatching.A recent study revealed the significance of the exosome in influenza A virus (IAV) ribogenesis and growth . In thisThe RNA exosome and its co-factors monitor the versatility and specificity of a huge variety of RNA substrates, and thus plays a crucial role in regulating the activity and maintaining the fidelity of gene expression. Numerous studies have revealed that an impaired RNA surveillance system can break RNA homeostasis, and thus cause detrimental consequences in multiple biological processes leading to human diseases . Howeve


The structure of the new compound was determined using a combination of spectroscopic techniques, including 1D and 2D NMR and electrospray-time of flight mass spectrometry (ESI-TOF MS). The relative configuration of compound 1, which contains two independent stereoclusters, has been established by molecular modelling in combination with nOe and coupling constant analyses. Biosynthetic arguments also allowed us to propose its absolute stereochemistry. The antimicrobial properties of the compounds isolated were evaluated against methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, Aspergillus fumigatus, and Candida albicans. The potent bioactivity previously reported for compounds 2 and 3 against methicillin-sensitive S. aureus has been extended to methicillin-resistant strains in this report.A new napyradiomycin, MDN-0170 ( Streptomycetaceae, which were first discovered from cultures of the actinomycete Chainia rubra chromene-5,10-dione, which was given the name MDN-0170 according to our proprietary compound library.The COSY spectrum of  1.82 H-1 ax and 11) was determined by analysis of key NOESY correlations  and the previously mentioned compounds must share a common biosynthetic route c 0.04, MeOH); IR (ATR) cm−1: 3350, 2955, 1712, 1680, 1618, 1265, 1082, 1017, 877, 779; (+)-ESI-TOFMS m/z 496.2080 [M + NH4]+ , 479.1818 [M + H]+ , 461.1721 [M + H − H2O]+ , 443.1614 [M + H − 2H2O]+ ; 1H and 13C NMR data see MDN-0170 , fungi (A. fumigatus ATCC46645), and yeast  following previously described methodologies [Compounds dologies ,27,28. Bdologies . In all 1–4 have been identified from the culture of Streptomyces sp. strain CA-271078, a Streptomyces strain related to Streptomyces aculeolatus NBRC 14824(T) which was isolated from an ascidian collected at the seaside in Baía Ana Chaves, Sao Tome (Sao Tome and Principe). Compounds 2 and 3 were able to inhibit strongly the growth of MRSA, one of the most common causes of hospital-acquired infections, with MIC values in the micromolar range. The activity found for compounds 2 and 3 against a clinical isolate of MRSA expands the panel of activity previously reported for these compounds against methicillin-sensitive S. aureus [13,20 seem to be essential for the biological activity of this structural class [1, containing two independent stereoclusters, has been established. Biosynthetic arguments for the compound also allowed us to propose its absolute stereochemistry. This article constitutes the first report on the chemical composition of extracts from a marine derived Streptomyces strain related to S. aculeolatus, and has identified this strain as a new source of milbemycins and NPDs related compounds. The research described herein also confirms that marine derived actinomycetes continue to be a rich and underexploited source of new small molecules that could lead to the discovery of new antibiotics.Compounds . aureus  to methial class ,6,12. Us


Cyanobacterium sp. IPPAS strain B-1200, isolated from Lake Balkhash, Kazakhstan, and characterized by the unique fatty acid composition of its membrane lipids, which are enriched with myristic and myristoleic acids. The approximate genome size is 3.4 Mb, and the predicted number of coding sequences is 3,119.Here, we report the draft genome of  Cyanobacterium sp. IPPAS B-1200 has been characterized by the unique fatty acid composition of its membrane lipids, which are enriched with myristic (30%), myristoleic (10%), palmitic (40%), and palmitoleic (10%) acids . It contained a total of 3,119 genes, with 2,934 genes coding for proteins, 137 pseudogenes, four rRNA-coding sequences, 40 tRNAs, and four noncoding RNAs. Two CRISPR arrays were found in the genome. The genome contained only one gene for the acyl-lipid Δ9-desaturase e lipids .KM502966 (16S rRNA sequence) and LWHC00000000 (PGAP file).Sequence data were deposited in GenBank under the following accession numbers: