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Introduction: Surgery and radiotherapy are key cancer treatments and the leading causes of damage to the lymphatics, a vascular network critical to fluid homeostasis and immunity. The clinical manifestation of this damage constitutes a devastating side-effect of cancer treatment, known as lymphoedema. Lymphoedema is a chronic condition evolving from the accumulation of interstitial fluid due to impaired drainage via the lymphatics and is recognised to contribute significant morbidity to patients who survive their cancer. Nevertheless, the molecular mechanisms underlying the damage inflicted on lymphatic vessels, and particularly the lymphatic endothelial cells (LEC) that constitute them, by these treatment modalities, remain poorly understood.Methods: We used a combination of cell based assays, biochemistry and animal models of lymphatic injury to examine the molecular mechanisms behind LEC injury and the subsequent effects on lymphatic vessels, particularly the role of the VEGF-C/VEGF-D/VEGFR-3 lymphangiogenic signalling pathway, in lymphatic injury underpinning the development of lymphoedema.Results: We demonstrate that radiotherapy selectively impairs key LEC functions needed for new lymphatic vessel growth (lymphangiogenesis). This effect is mediated by attenuation of VEGFR-3 signalling and downstream signalling cascades. VEGFR-3 protein levels were downregulated in LEC that were exposed to radiation, and LEC were therefore selectively less responsive to VEGF-C and VEGF-D. These findings were validated in our animal models of radiation and surgical injury.Discussion: Our data provide mechanistic insight into injury sustained by LEC and lymphatics during surgical and radiotherapy cancer treatments and underscore the need for alternative non-VEGF-C/VEGFR-3-based therapies to treat lymphoedema.

Compact reduced graphene oxide powder was prepared via Li+-assisted treatment of graphene oxide and exhibited a high specific volume capacitance.

Like other viruses, SARS-CoV-2 causes different symptoms and different degrees of harmfulness to different individuals. Potential reasons include an individual’s viral dose exposure, the affinity of an individual’s ACE2 to the spike protein of the virus, and the ability of the individual’s induced immune system to neutralize the virus. Beyond these, an individual’s epitranscriptomic system could be among the causes as well. The viral RNA genome, once inside the host cell, can be subject to modifications by the host’s epitranscriptomic machinery. Because the machinery is different in different individuals, it is reasonable to believe that RNA modifications are different among different individuals, and can positively or negatively affect downstream events that involve the RNA such as replication of viral genome, generation of viral mRNAs, viral protein production, RNA recognition by host’s immune system, and packaging of RNA genome into new viral particles. In this context, we studied the effects of several RNA modifications including pseudouridine (Ψ), 5-methylcytosine (m5C), N6-methyladenosine (m6A), N1-methyladenosine (m1A) and N3-methylcytosine (m3C) on the catalytic activity of SARS-CoV-2 replication complex (SC2RC), which included RNA dependent RNA polymerase (RdRp). We found that Ψ, m5C, m6A and m3C had little effects on the activity, while m1A severely inhibited the enzyme. Both m1A and m3C disrupt canonical base pairing. It is interesting one of them inhibits the enzyme while the other does not. The fact that m1A inhibits SC2RC may imply that the modification can be difficult to identify using any method even though it may exist and play a critical role. Putting aside other mechanisms by which the modifications cause individualized symptoms, the results indicated that individuals with a higher chance of m1A modification may stop viral replication and have less severe symptoms. However, this contradicts the observations that individuals with clinical conditions such as cancer, obesity and diabetes, who have upregulated m1A modifications, are more vulnerable to COVID-19. This contradiction may be explained by the importance of the dynamic nature of epitranscriptomic modifications for viral survival.

The design of microstructures and the optimum selection of electrode materials have substantial effects on the electrochemical performances of supercapacitors. A core–shell structured CuCo2S4@Ni(OH)2 electrode material was designed, with CuCo2S4 nanotubes as the core wrapped by interlaced Ni(OH)2 nano-sheets as the shell. The hydrothermal and electro-deposition processes were adopted to synthesize CuCo2S4@Ni(OH)2 materials. The CuCo2S4 nanotubes can both provide specific capacitance and act as a “superhighway” for electrons due to their highly conductive skeleton structure. The Ni(OH)2 nano-sheets will boost the electrochemically active sites and enhance the specific surface area. Meanwhile, the mutually restricted core–shell CuCo2S4@Ni(OH)2 electrode could regulate the volume deformation to improve its stability. The CuCo2S4@Ni(OH)2 electrode had a maximum specific capacitance of 2668.4 F g−1 at a current density of 1 A g−1 and a superior cycling stability of 90.3% after 10 000 cycles. Moreover, a CuCo2S4@Ni(OH)2//active carbon asymmetric supercapacitor with a maximum energy density of 44 W h kg−1 was assembled, suggesting that CuCo2S4@Ni(OH)2 is a successful binder-free electrode material for high performance supercapacitors.

On the basis of a previously identified DOT1L peptide mimetic (compound

Reduced graphene oxide (RGO) with a porous nanostructure and large surface area suffers from poor volumetric capacitance when it is employed in supercapacitors. This paper reports a facile, scalable and efficient method to produce self-assembled reduced graphene oxide with high packing density (1.55 g cm−3) via phosphoric acid treatment (PAT) at moderate temperatures (200–400 °C). The PAT process could not only controllably preserve redox-active C–OH and CO groups which can provide large and stable pseudocapacitance, but also contribute to the formation of a well self-assembled compact structure. Due to the pseudocapacitive behavior of the electrochemically active oxygen functional groups as well as the compact layered structure, the reduced graphene oxide (RGO-MP40) exhibits an ultrahigh volumetric capacitance (482 F cm−3 and 384 F cm−3 at a mass loading of 2.5 mg cm−2 and 19.7 mg cm−2, respectively). This novel strategy holds great promise for designing graphene-based materials for compact energy-storage devices.

A high-shear mixer was used in the process of chemical precipitation to prepare graphene oxide (GO) and Ni(OH)2 composites with different weight fractions of GO from 0 wt% to 2.6 wt%. The GO/Ni(OH)2 composite with 1.5 wt% GO prepared by high-shear mixing (1.5GO/Ni(OH)2–H) displayed a high specific capacitance (1836 F g−1 at 2 mV s−1), which was beyond that (1581 F g−1 at 2 mV s−1) of GO/Ni(OH)2 obtained by impeller agitator stirring (1.5GO/Ni(OH)2–M). 1.5GO/Ni(OH)2–H also showed excellent cycling stability, retaining 91.7% of its initial specific capacitance after 2000 cycles. Furthermore, the asymmetric device (the 1.5GO/Ni(OH)2–H as the positive electrode and active carbon as the negative electrode) exhibited a high energy density of 41.2 W h kg−1 at a power density of 1500 W kg−1 with a voltage of 1.5 V. In addition, the effects of high-shear mixing and impeller agitator stirring on the microstructures and electrochemical properties of the composites were discussed, which indicated that high-shear mixing could exfoliate graphene oxide and restrain the grain growth of Ni(OH)2, thus leading to excellent electrochemical performance.

The design of microstructures and the optimum selection of electrode materials have substantial effects on the electrochemical performances of supercapacitors. A core-shell structured CuCo2S4@Ni(OH)2 electrode material was designed, with CuCo2S4 nanotubes as the core wrapped by interlaced Ni(OH)2 nano-sheets as the shell. The hydrothermal and electro-deposition processes were adopted to synthesize CuCo2S4@Ni(OH)2 materials. The CuCo2S4 nanotubes can both provide specific capacitance and act as a "superhighway" for electrons due to their highly conductive skeleton structure. The Ni(OH)2 nano-sheets will boost the electrochemically active sites and enhance the specific surface area. Meanwhile, the mutually restricted core-shell CuCo2S4@Ni(OH)2 electrode could regulate the volume deformation to improve its stability. The CuCo2S4@Ni(OH)2 electrode had a maximum specific capacitance of 2668.4 F g-1 at a current density of 1 A g-1 and a superior cycling stability of 90.3% after 10 000 cycles. Moreover, a CuCo2S4@Ni(OH)2//active carbon asymmetric supercapacitor with a maximum energy density of 44 W h kg-1 was assembled, suggesting that CuCo2S4@Ni(OH)2 is a successful binder-free electrode material for high performance supercapacitors.

This protocol provides details for the preparation of nucleoside phosphoramidites with 1,3-dithian-2-yl-methyl (Dim) and 1,3-dithian-2-yl-methoxycarbonyl (Dmoc) as protecting groups, and a linker with Dmoc as the cleavable function, then using them for solid phase synthesis of sensitive oligodeoxynucleotides (ODNs). Using these Dim-Dmoc phosphoramidites and Dmoc linker, ODN synthesis can be achieved under typical conditions using phosphoramidite chemistry with slight modifications, and ODN deprotection and cleavage can be achieved under mild conditions involving oxidation with sodium periodate at pH 4 followed by aniline at pH 8. Under the mild deprotection and cleavage conditions, many sensitive functional groups including but not limited to esters, thioesters, alkyl halides, N-aryl amides, and α-chloroamides-which cannot survive the basic and nucleophilic deprotection and cleavage conditions such as concentrated ammonium hydroxide and dilute potassium methoxide used in typical ODN synthesis technologies-can survive. Thus, it is expected that the Dim-Dmoc ODN synthesis technology will find applications in the synthesis of ODNs that contain a wide range of sensitive functional groups. © 2020 Wiley Periodicals LLC. Basic Protocol: Synthesis, deprotection, cleavage, and purification of sensitive oligodeoxynucleotides Support Protocol 1: Synthesis of Dim-Dmoc nucleoside phosphoramidites Support Protocol 2: Preparation of CPG with a Dmoc linker Support Protocol 3: Synthesis of a phosphoramidite containing a sensitive alkyl ester group.

Fat grafting is a well-established surgical technique used in plastic surgery to restore deficient tissue, and more recently, for its putative regenerative properties. Despite more frequent use of fat grafting, however, a scientific understanding of the mechanisms underlying either survival or remedial benefits of grafted fat remain lacking. Clinical use of fat grafts for breast reconstruction in tissues damaged by radiotherapy first provided clues regarding the clinical potential of stem cells to drive tissue regeneration. Healthy fat introduced into irradiated tissues appeared to reverse radiation injury (fibrosis, scarring, contracture and pain) clinically; a phenomenon since validated in several animal studies. In the quest to explain and enhance these therapeutic effects, adipose-derived stem cells (ADSCs) were suggested as playing a key role and techniques to enrich ADSCs in fat, in turn, followed. Stem cells - the body’s rapid response ‘road repair crew’ - are on standby to combat tissue insults. ADSCs may exert influences either by releasing paracrine-signalling factors alone or as cell-free extracellular vesicles (EVs, exosomes). Alternatively, ADSCs may augment vital immune/inflammatory processes; or themselves differentiate into mature adipose cells to provide the ‘building-blocks’ for engineered tissue. Regardless, adipose tissue constitutes an ideal source for mesenchymal stem cells for therapeutic application, due to ease of harvest and processing; and a relative abundance of adipose tissue in most patients. Here, we review the clinical applications of fat grafting, ADSC-enhanced fat graft, fat stem cell therapy; and the latest evolution of EVs and nanoparticles in healing, cancer and neurodegenerative and multiorgan disease.

A high-shear mixer was used in the process of chemical precipitation to prepare graphene oxide (GO) and Ni(OH)2 composites with different weight fractions of GO from 0 wt% to 2.6 wt%. The GO/Ni(OH)2 composite with 1.5 wt% GO prepared by high-shear mixing (1.5GO/Ni(OH)2-H) displayed a high specific capacitance (1836 F g-1 at 2 mV s-1), which was beyond that (1581 F g-1 at 2 mV s-1) of GO/Ni(OH)2 obtained by impeller agitator stirring (1.5GO/Ni(OH)2-M). 1.5GO/Ni(OH)2-H also showed excellent cycling stability, retaining 91.7% of its initial specific capacitance after 2000 cycles. Furthermore, the asymmetric device (the 1.5GO/Ni(OH)2-H as the positive electrode and active carbon as the negative electrode) exhibited a high energy density of 41.2 W h kg-1 at a power density of 1500 W kg-1 with a voltage of 1.5 V. In addition, the effects of high-shear mixing and impeller agitator stirring on the microstructures and electrochemical properties of the composites were discussed, which indicated that high-shear mixing could exfoliate graphene oxide and restrain the grain growth of Ni(OH)2, thus leading to excellent electrochemical performance.

The variation of oxygen functional groups (OFGs) is extremely complicated during the thermal treatment of graphene oxide (GO). Selectively improving the content of electrochemically active OFGs through multi-step thermal treatment is a promising strategy to enhance the charge storage capability of graphene-based supercapacitor electrodes. In this work, a two-step thermal treatment method was used to produce reduced graphene oxide (RGO) with selectively increasing the content of electrochemically active quinone-type carbonyl groups (C˭O). The first step is thermal reduction at 500 °C in Ar atmosphere, during which GO is exfoliated into separated sheets with porous structure and larger surface area; the second step is air oxidation at 400 °C, which facilitates the selective formation of quinone-type C˭O groups and enhances the mesoporous fraction. Due to the pseudocapacitive behavior of quinone-type C˭O groups and low ion diffusion resistance, the electrode prepared by RGO exhibits a high specific capacitance of 303 F g−1 at 0.2 A g−1 with excellent rate capability (202 F g−1 at 10 A g−1) and notable cycling performance (~ 116% capacitance retention after 1500 cycling at 1 A g−1). The present work proposed a facile and environmentally friendly two-step thermal treatment strategy in effectively controlling the surface property and microstructure of graphene materials, which has significant implications for mass producing graphene materials for supercapacitor electrodes.

The debilitating condition known as secondary lymphedema frequently occurs after lymphadenectomy and/or radiotherapy for the treatment of cancer. These therapies can damage lymphatic vessels leading to edema, fibrosis, inflammation and dysregulated adipogenesis, which result in profound swelling of an affected limb. Importantly, lymphedema patients often exhibit impaired immune function which predisposes them to a variety of infections. It is known that lymphadenectomy can compromise the acquisition of adaptive immune responses and antibody production; however the cellular mechanisms involved are poorly understood. Here we discuss recent progress in revealing the cellular and molecular mechanisms underlying poor immune function in secondary lymphedema, which has indicated a key role for regulatory T cells in immunosuppression in this disease. Furthermore, the interaction of CD4+ T cells and macrophages has been shown to play a role in driving proliferation of lymphatic endothelial cells and aberrant lymphangiogenesis, which contribute to interstitial fluid accumulation in lymphedema. These new insights into the interplay between lymphatic vessels and the immune system in lymphedema will likely provide opportunities for novel therapeutic approaches designed to improve clinical outcomes in this problematic disease.

Lung cancer, especially non-small-cell lung cancer (NSCLC), plays the leading role in cancer which is closely related to a myriad of fatal results. Unfortunately, current molecular mechanisms and clinical treatment of NSCLC still remain to be explored despite the fact that intensive investigations have been carried out in the last two decades. Recently, growing attention to finding exploitable sources of anticancer agents is refocused on quinolone compounds, an antibiotic with a long period of clinic application, for their remarkable cell-killing activity against not only bacteria, but eukaryotes as well. In this study, we found LZ-106, an analog of enoxacin, exhibiting potent inhibitory effects on NSCLC in both cultured cells and xenograft mouse model. We identified apoptosis-inducing action of LZ-106 in NSCLC cells through the mitochondrial and endoplasmic reticulum (ER)-stress apoptotic pathways via Annexin-V/PI double-staining assay, membrane potential detection, calcium level detection and the expression analysis of the key apoptotic proteins. Through comet assay, reactive oxygen species (ROS) detection, the expression analysis of DNA damage response (DDR) marker γ-H2AX and other DDR-related proteins, we also demonstrated that LZ-106 notably induced ROS overproduction and DDR. Interestingly, additional evidence in our findings revealed that DDR and apoptosis could be alleviated in the presence of ROS scavenger N-acetyl-cysteine (NAC), indicating ROS-dependent DDR involvement in LZ-106-induced apoptosis. Thus our data not only offered a new therapeutic candidate for NSCLC, but also put new insights into the pharmacological research of quinolones.

Leakage of fluid from blood vessels, leading to oedema, is a key feature of many diseases including hyperoxic acute lung injury (HALI), which can occur when patients are ventilated with high concentrations of oxygen (hyperoxia). The molecular mechanisms driving vascular leak and oedema in HALI are poorly understood. VEGF-D is a protein that promotes blood vessel leak and oedema when overexpressed in tissues, but the role of endogenous VEGF-D in pathological oedema was unknown. To address these issues, we exposed Vegfd-deficient mice to hyperoxia. The resulting pulmonary oedema in Vegfd-deficient mice was substantially reduced compared to wild-type, as was the protein content of bronchoalveolar lavage fluid, consistent with reduced vascular leak. Vegf-d and its receptor Vegfr-3 were more highly expressed in lungs of hyperoxic, versus normoxic, wild-type mice, indicating that components of the Vegf-d signalling pathway are up-regulated in hyperoxia. Importantly, VEGF-D and its receptors were co-localized on blood vessels in clinical samples of human lungs exposed to hyperoxia; hence, VEGF-D may act directly on blood vessels to promote fluid leak. Our studies show that Vegf-d promotes oedema in response to hyperoxia in mice and support the hypothesis that VEGF-D signalling promotes vascular leak in human HALI. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

In this study, a new, effective strategy is reported for the fabrication of composites using manganese oxide (MnO2) grown in situ on three-dimensional (3D) graphene by the reverse microemulsion (water-in-oil) method. A uniform coating of nanoscale MnO2 layers can be observed on the internal surface of 3D graphene, which could benefit rapid ionic and electronic transport. The electrochemical performance of the MnO2/3D graphene composites is optimized by the control of the composite structure and mass loading of MnO2. The MnO2/3D graphene composite thus prepared exhibits a significantly high specific capacitance of 659.7 F g−1 at 0.3 A g−1 and an excellent retention life of 106% after 1000 cycles. The facile synthesis and excellent electrochemical performance of the MnO2/3D graphene composites indicate that the developed method demonstrates potential applications for the fabrication of novel electrode materials for use in energy storage devices.

Manganese oxide (MnO2)/three-dimensional (3D) reduced graphene oxide (RGO) composites were prepared by a reverse microemulsion (water/oil) method. MnO2 nanoparticles (3–20 nm in diameter) with different morphologies were produced and dispersed homogeneously on the macropore surfaces of the 3D RGO. Scanning electron microscopy and transmission electron microscopy were applied to characterize the microstructure of the composites. The MnO2/3D RGO composites, which were annealed at 150 °C, displayed a significantly high specific capacitance of 709.8 F g−1 at 0.2 A g−1. After 1000 cycles, the capacitance retention was measured to be 97.6%, which indicates an excellent long-term stability of the MnO2/3D RGO composites.

Herein, graphene oxide (GO) with a 3D structure was prepared on the surface of Ni foam (NF) via electrophoretic deposition, and then reacted in situ to form 3D reduced graphene oxide (RGO) via thermal reduction. Ni–Co–S nanosheet arrays were produced on various substrates (RGO/NF, GO/NF and NF) via a facile one-step electrochemical deposition. Scanning electron microscopy (SEM) demonstrated that RGO nanosheets were vertically wrapped on NF by thermal reduction of GO. Furthermore, interconnected and hierarchical porous Ni–Co–S nanosheets were uniformly coated on RGO. The electrochemical performance of the ternary material on different substrates was investigated. The physical structures, combined with the advantages of both ternary Ni–Co–S and RGO, exhibited excellent electrochemical performance. When incorporated as electrode materials for supercapacitors, the synthesized samples possess high specific capacitance and a long cycle life. With the synergistic effect of RGO and ternary Ni–Co–S, a high performance is achieved. The specific capacitance of Ni–Co–S/RGO/NF (2643 F g−1) demonstrated an enhancement compared with Ni–Co–S/GO/NF (2083 F g−1) and Ni–Co–S/NF (1329 F g−1) at a current density of 10 A g−1. Additionally, the retention of specific capacitance of Ni–Co–S/RGO/NF after 2500 cycles displayed a superior cyclic stability of 84.22% at a current density of 50 A g−1.

Antisense transcription is widespread in many genomes and plays important regulatory roles in gene expression. The objective of our study was to investigate the extent and functional relevance of antisense transcription in forest trees. We employed Populus, a model tree species, to probe the antisense transcriptional response of tree genome under drought, through stranded RNA-seq analysis. We detected nearly 48% of annotated Populus gene loci with antisense transcripts and 44% of them with co-transcription from both DNA strands. Global distribution of reads pattern across annotated gene regions uncovered that antisense transcription was enriched in untranslated regions while sense reads were predominantly mapped in coding exons. We further detected 1185 drought-responsive sense and antisense gene loci and identified a strong positive correlation between the expression of antisense and sense transcripts. Additionally, we assessed the antisense expression in introns and found a strong correlation between intronic expression and exonic expression, confirming antisense transcription of introns contributes to transcriptional activity of Populus genome under drought. Finally, we functionally characterized drought-responsive sense–antisense transcript pairs through gene ontology analysis and discovered that functional groups including transcription factors and histones were concordantly regulated at both sense and antisense transcriptional level. Overall, our study demonstrated the extensive occurrence of antisense transcripts of Populus genes under drought and provided insights into genome structure, regulation pattern and functional significance of drought-responsive antisense genes in forest trees. Datasets generated in this study serve as a foundation for future genetic analysis to improve our understanding of gene regulation by antisense transcription.

Denaturing reversed-phase (RP) high performance liquid chromatography (HPLC) is usually achieved by elevating column temperature. In this article, an alternative method involving using a mobile phase that contains urea and performing HPLC at room temperature is described. The efficacy of the new method was demonstrated by analyzing a 61-mer oligodeoxynucleotide (ODN) and double-stranded (ds) ODNs. The multiple peaks of the 61-mer ODN under normal conditions merged into one under the denaturing conditions. The broad single peaks of dsODNs under normal conditions were split into two sharp peaks.

Oligodeoxynucleotide (ODN) purification was achieved by capping failure sequences with a polymerizable methacrylamide phosphoramidite during automated synthesis, polymerizing the failure sequences into an acrylamide gel after cleavage and deprotection, and extraction of full-length sequences with water. The details regarding the technology including the capping efficiency of four polymerizable phosphoramidites, optimal capping time, diffusion speeds of ODN from gels with different cross-linking ratios to solution, and the efficiency of ODN extraction from gel were investigated. In addition, the technology was tested for purification of a long sequence and purification on larger scales. We also found that polymerization of failure sequences in a centrifuge tube in air did not affect purification results. Finally, we provided additional evidence that ODNs are stable under radical polymerization conditions by complete digestion of ODN followed by reversed-phase HPLC analysis of nucleosides.

Eragrostis tef (Zucc.), a member of the Chloridoideae subfamily of grasses, is one of the most important food crops in Ethiopia. Lodging is the most important production problem in tef. The rht1 and sd1 dwarfing genes have been useful for improving lodging resistance in wheat and rice, respectively, in what has been known as the “Green Revolution.” All homologs of rht1 and sd1 were cloned and sequenced from 31 tef accessions collected from across Ethiopia. The allotetraploid tef genome was found to carry two rht1 homologs. From sequence variation between these two putative homologs, an approximate ancestral divergence date of 6.4 million years ago was calculated for the two genomes within tef. Three sd1 homologs were identified in tef, with unknown orthologous/paralogous relationships. The genetic diversity in the 31 studied accessions was organized into a relatively small number of haplotypes (2−4) for four of these genes, whereas one rht1 homeologue exhibited 10 haplotypes. A low level of nucleotide diversity was observed at all loci. Linkage disequilibrium analysis demonstrated strong linkage disequilibrium, extending the length of the five genes investigated (2−4 kb), with no significant decline. There was no significant correlation between haplotypes of any of these genes and their recorded site of origin.

Background/Aims: Prostate cancer runs in families and shows a clear dietary involvement. Until recently, the key risk gene(s) have proved elusive. We summarise current understandings of nutrient-gene interactions in prostate cancer risk and progression. Methods: A MEDLINE-based literature search was conducted. Results: Hypothesis-directed candidate gene approaches provide plausible, albeit statistically weak, nutrient-gene interactions. These are based on early understandings of factors likely to impact on carcinogenesis, including both nutrient and genetic effects on androgen biosynthesis and action, xenobiotic metabolism, DNA damage and DNA repair. Non-hypothesis-directed genome-wide association studies provide much stronger evidence for other genes, not hitherto suspected for involvement. Although only a few of these have been formally tested for dietary associations in well-designed epidemiologic studies, the nature of many of the genes suggests that their activity may be regulated by nutrients. These effects may not only be relevant to prostate cancer susceptibility, but also to disease progression. Conclusions: It will be important to move beyond studying single nucleotide polymorphisms, into more complex chromosomal rearrangements and to epigenetic changes. For future progress, large international cohorts will not only need to provide proof of individual nutrient-gene interactions, but also to relate these to more complex nutrient-gene-gene interactions, as parts of pathways. Bioinformatics and biostatistics will be increasingly important tools in nutrigenetic studies beyond 2011.

Isochorismate synthase (ICS) converts chorismate to isochorismate for the biosynthesis of phylloquinone, an essential cofactor for photosynthetic electron transport. ICS is also required for salicylic acid (SA) synthesis during Arabidopsis defense. In several other species, including Populus , SA is derived primarily from the phenylpropanoid pathway. We therefore sought to investigate ICS regulation in Populus to learn the extent of ICS involvement in SA synthesis and defense. Arabidopsis harbors duplicated AtICS genes that differ in their exon-intron structure, basal expression, and stress inducibility. In contrast, we found a single ICS gene in Populus and six other sequenced plant genomes, pointing to the AtICS duplication as a lineage-specific event. The Populus ICS encodes a functional plastidic enzyme, and was not responsive to stresses that stimulated phenylpropanoid accumulation. Populus ICS underwent extensive alternative splicing that was rare for the duplicated AtICSs . Sequencing of 184 RT-PCR Populus clones revealed 37 alternative splice variants, with normal transcripts representing ≈50% of the population. When expressed in Arabidopsis , Populus ICS again underwent alternative splicing, but did not produce normal transcripts to complement AtICS1 function. The splice-site sequences of Populus ICS are unusual, suggesting a causal link between junction sequence, alternative splicing, and ICS function. We propose that gene duplication and alternative splicing of ICS evolved independently in Arabidopsis and Populus in accordance with their distinct defense strategies. AtICS1 represents a divergent isoform for inducible SA synthesis during defense. Populus ICS primarily functions in phylloquinone biosynthesis, a process that can be sustained at low ICS transcript levels.

Salicin-based phenolic glycosides, hydroxycinnamate derivatives and flavonoid-derived condensed tannins comprise up to one-third of Populus leaf dry mass. Genes regulating the abundance and chemical diversity of these substances have not been comprehensively analysed in tree species exhibiting this metabolically demanding level of phenolic metabolism. Here, shikimate-phenylpropanoid pathway genes thought to give rise to these phenolic products were annotated from the Populus genome, their expression assessed by semiquantitative or quantitative reverse transcription polymerase chain reaction (PCR), and metabolic evidence for function presented. Unlike Arabidopsis, Populus leaves accumulate an array of hydroxycinnamoyl-quinate esters, which is consistent with broadened function of the expanded hydroxycinnamoyl-CoA transferase gene family. Greater flavonoid pathway diversity is also represented, and flavonoid gene families are larger. Consistent with expanded pathway function, most of these genes were upregulated during wound-stimulated condensed tannin synthesis in leaves. The suite of Populus genes regulating phenylpropanoid product accumulation should have important application in managing phenolic carbon pools in relation to climate change and global carbon cycling.

A new technology was developed to assist gene-enrichment sequencing of any complex plant genome, employing maize as the test organism. Hypomethylated partial restriction (HMPR) libraries were constructed by using independent partial digestions with methylation-sensitive restriction enzymes HpaII (5′-CCGG-3′) and HpyCH4IV (5′-ACGT-3′). Fragments of 1-4 kb were purified and cloned, followed by sequence analysis of >2000 clones from 10 separate libraries. Organellar clones comprised ∼10% of each library but were useful in showing that no chimeric clones were generated and that digestion efficiencies were 10%-25% in different libraries. Four separate HMPR libraries, analyzed in detail, exhibited very similar degrees of gene enrichment and repeat depletion. Known gene homologies were found in ∼25% of the HMPR clones, compared with 70% for unfiltered maize sequences) and in providing an unmatched enrichment for the “unknown” sequences that contain promoters, introns, and other gene-adjacent regions.

Little is known about the physical makeup of heterochromatin in the soybean (Glycine max L. Merr.) genome. Using DNA sequencing and molecular cytogenetics, an initial analysis of the repetitive fraction of the soybean genome is presented. BAC 076J21, derived from linkage group L, has sequences conserved in the pericentromeric heterochromatin of all 20 chromosomes. FISH analysis of this BAC and three subclones on pachytene chromosomes revealed relatively strict partitioning of the heterochromatic and euchromatic regions. Sequence analysis showed that this BAC consists primarily of repetitive sequences such as a 102-bp tandem repeat with sequence identity to a previously characterized ∼120-bp repeat (STR120). Fragments of Calypso-like retroelements, a recently inserted SIRE1 element, and a SIRE1 solo LTR were present within this BAC. Some of these sequences are methylated and are not conserved outside of G. max and G. soja, a close relative of soybean, except for STR102, which hybridized to a restriction fragment from G. latifolia. These data present a picture of the repetitive fraction of the soybean genome that is highly concentrated in the pericentromeric regions, consisting of rapidly evolving tandem repeats with interspersed retroelements.

Higher eukaryotic genomes, including those from plants, contain large amounts of repetitive DNA that complicate genome analysis. We have developed a technique based on DNA renaturation which normalizes repetitive DNA, and thereby allows a more efficient outcome for full genome shotgun sequencing. The data indicate that sequencing the unrenatured outcome of a Cot experiment, otherwise known as High-Cot DNA, enriches genic sequences by more than fourfold in maize, from 5% for a random library to more than 20% for a High-Cot library. Using this approach, we predict that gene discovery would be greater than 95% and that the number of sequencing runs required to sequence the full gene space in maize would be at least fourfold lower than that required for full-genome shotgun sequencing.

The resistance of tomato to the pathogenic fungus Cladosporiumfulvum complies with the gene-for-gene relationship. Race specificresistance is based on Cf-gene mediated recognition ofsecreted avirulence products, resulting in a hypersensitive response (HR).Besides the avirulence gene products, C. fulvum secretes anumber of extra cellular proteins (ECPs) into the apoplast. Two L.esculentum accessions have previously been identified that reactedwith a HR upon injection with purified ECP3. The corresponding resistance genedesignated Cf-ECP3 was mapped by using an F2population composed of 192 plants from the cross of susceptible MoneyMaker toresistant L. esculentum G1.1153.Cf-ECP3 inherited monogenically, cosegragated with theChromosome 1 Cleaved Amplified Polymorphic Sequence (CAPS) marker CT116 and wasmapped accurately at Orion, a locus harbouring Cf-ECP2 inother genotypes. RFLP anaysis with a Cf-9 probe furtherdemonstrated cosegregation of Cf-ECP3 with anHcr9 (Homologue of Cladosporiumfulvumresistance gene Cf-9) indicating that this gene is likelyan Hcr9. Thus in addition to the Milky Way locusharbouringthe Cf-4, Cf-4A andCf-9 resistance genes targeted against AVR4, AVR4A andAVR9, Orion is another complex locus on the short arm of Chromosome 1 thatharbours at least two functional Cf-genes,Cf-ECP2 and Cf-ECP3, targeted againstthe fungal excreted proteins ECP2 and ECP3.

Salicylic acid (SA) has long been implicated in plant responses to oxidative stress. SA overproduction in Arabidopsis thaliana leads to dwarfism, making in planta assessment of SA effects difficult in this model system. We report that transgenic Populus tremula × alba expressing a bacterial SA synthase hyperaccumulated SA and SA conjugates without negative growth consequences. In the absence of stress, endogenously elevated SA elicited widespread metabolic and transcriptional changes that resembled those of wild-type plants exposed to oxidative stress-promoting heat treatments. Potential signaling and oxidative stress markers azelaic and gluconic acids as well as antioxidant chlorogenic acids were strongly coregulated with SA, while soluble sugars and other phenylpropanoids were inversely correlated. Photosynthetic responses to heat were attenuated in SA-overproducing plants. Network analysis identified potential drivers of SA-mediated transcriptome rewiring, including receptor-like kinases and WRKY transcription factors. Orthologs of Arabidopsis SA signaling components NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES1 and thioredoxins were not represented. However, all members of the expanded Populus nucleoredoxin-1 family exhibited increased expression and increased network connectivity in SA-overproducing Populus, suggesting a previously undescribed role in SA-mediated redox regulation. The SA response in Populus involved a reprogramming of carbon uptake and partitioning during stress that is compatible with constitutive chemical defense and sustained growth, contrasting with the SA response in Arabidopsis, which is transient and compromises growth if sustained.

This unit describes the purification of synthetic oligodeoxyribonucleotides (ODN) using a catching-by-polymerization approach. In a crude ODN, the major impurity is the failure sequences generated in the coupling step of each synthetic cycle. They are difficult to remove due to the similarity of their physical properties to the full-length sequences. Two non-chromatographic methods are described in the unit to solve the problem. In the first one, during automated synthesis, the failure sequences are tagged with a methacrylamide group, which is polymerizable and can participate in acrylamide radical polymerization reactions; the full-length sequences are not tagged. After synthesis, the crude mixture is subjected to polymerization. The failure sequences are incorporated into an insoluble polymer; the full-length sequences are extracted with water. In the second method, the full-length sequences are tagged with a methacrylamide group via a cleavable linker; the failure sequences are not tagged. After synthesis, the full-length sequences are incorporated into a polymer; the failure sequences are washed away with water. Pure full-length sequences are cleaved from the polymer. The two methods are complementary. Curr. Protoc. Nucleic Acid Chem. 49:10.14.1-10.14.21. © 2012 by John Wiley & Sons, Inc. Keywords: oligonucleotide; purification; catching by polymerization; scalable purification; acrylamide radical polymerization; full-length sequence; failure sequence

Phenolic metabolites that contain salicylic acid (SA)-like moieties are major non-structural constituents in Populus leaves, shoots and roots. These so-called phenolic glycosides (PGs) are taxonomically limited to the Salicaceae, where they are known to mitigate insect and animal herbivory. SA itself is an important signaling molecule in plant defense and abiotic stress responses, and is derived from the isochorismate or phenylpropanoid pathways, depending on the species [1,2]. Hyper-accumulation of SA reduces growth in Arabidopsis[3], while high levels of PG have been associated with reduced growth in Populus[4]. Although the common PGs, salicin, salicortin, and their derivatives contain a salicyl moiety, a direct metabolic relationship between PG and SA in Populus has not been shown. We have therefore been using stable isotope tracers, cell culture feeding, functional genomics and transgenic perturbation to probe the relationship between SA, PG and growth in Populus. Stable isotope incorporation confirmed a hydroxycinnamate-benzoate origin of the salicyl moiety of PGs, and provided the first empirical evidence that the bioactive hydroxycyclohexenone moiety of salicortin is also a phenylpropanoid derivative [5]. Therefore it is unlikely that PG biosynthesis involves the salicylic acid pathway. To further address any possible involvement of the isochorismate pathway in PG biosynthesis, isochorismate synthase (ICS) was characterized. ICS is present as a single-gene in most sequenced plant genomes, but is duplicated in Arabidopsis. In Arabidopsis, chorismate-derived SA is the obligatory route for defense signaling, mediated by AtICS1 in response to various biotic and abiotic cues. In contrast, the single-copy Populus ICS is not stress-inducible, and is involved in biosynthesis of phylloquinone for photosynthetic electron transport [2]. We found no evidence of ICS involvement in SA or PG biosynthesis in transgenic Populus, pointing to lineage-specific evolution of the ICS-derived SA pathway in Arabidopsis. We transformed Populus with the bacterial genes for the biosynthesis and degradation of SA for a more in-depth investigation of the possibility of an SA interaction with PG regulation. SA-hyperaccumulating and SA-deficient lines were generated by expressing a salicylate synthase and a salicylate hydroxylase, respectively. SA was converted to SA-glycoside (SAG) and gentisic acid-glucoside (GAG) in the hyperaccumulating lines. SAG and GAG were very low in wild-type and were not detected in the SA-deficient lines. Despite these clear differences, no changes in PG levels were detected. These results argue against SA as potential precursor of PGs. Survival and establishment of rooted cuttings were negatively affected by SA-hyperaccumulation. Once established, however, growth rates were similar among plant lines, in sharp contrast to SA-over-producing Arabidopsis. SA-hyperaccumulating lines exhibited altered thermal tolerance, based on electrolyte leakage assays and metabolite profiling. Together, our results provide support for a phenylpropanoid origin of PGs, and argue against the involvement of SA in PG biosynthesis. The data is consistent with complementary roles for SA and PG in Populus fitness. A number of other experiments were conducted to investigate the regulation of PG homeostasis in Populus. Administration of a putative PG precursor salicyl alcohol to cell cultures yielded the glucoside, salicin. Salicyl alcohol-feeding also altered the partitioning of carbon into condensed tannin (CT) biosynthesis, another quantitatively important phenylpropanoid product [6]. Salicyl alcohol-feeding induced expression of several glycosyltransferases (GT1), as well as genes associated with sucrose catabolism, glycolysis and the Krebs cycle, presumably to accommodate the increased glycosylation. Conversely, transcript levels of most of the flavonoid pathway genes were reduced, consistent with reduced CT synthesis. Given that glycosylation was the only PG biosynthetic step occurring in the fed-cells, the results suggest that glycosylation of phenolic products can play a role in regulating the tradeoff between competing chemical defenses, e.g., PGs and CTs. Correlation analysis was used to identify PG-coregulated genes from a number of inductive treatments. One such candidate, SUT4 encoding a tonoplast sucrose transporter, was of particular interest because of the reported salicin transport activity of its Arabidopsis ortholog. RNAi-silencing of SUT4 altered sucrose distribution between source and sink organs, and reduced PG accrual by 20-30%, accompanied by altered biomass partitioning [7]. Chemical and molecular analyses revealed a complex network linking PG accrual and carbohydrate homeostasis. PG-coregulated GT1 members were also targeted for characterization, due to the prominent role of glycosylation in PG biosynthesis. We identified a novel salicyl alcohol glycosyltransferase (UGT71L1). Over-expression of UGT71L1 in transgenic Populus led to a ~2-fold increase in concentrations of salicin and salicortin in roots. The quantitative effect on secondary metabolism is significant, as PGs are the second most abundant non-structural phenolics, after CTs, in the roots of this poplar clone. UGT71L1 is phylogenetically distinct from the well-characterized SAGTs. It belongs to a Populus-specific clade with no close homologs in Arabidopsis, Vitis,Medicago and Oryza, consistent with the limited taxonomic range of PGs. Overall, the experiments described highlight the important contributions of sugar transport and phenolic glycosylation to PG homeostasis, while also showing that SA and PGs make independent but complementary contributions toward the maintenance of Populus fitness.

Maize is a staple crop of the grass family and also an excellent model for plant genetics. Owing to the large size and repetitiveness of its genome, we previously investigated two approaches to accelerate gene discovery and genome analysis in maize: methylation filtration and high C(0)t selection. These techniques allow the construction of gene-enriched genomic libraries by minimizing repeat sequences due to either their methylation status or their copy number, yielding a 7-fold enrichment in genic sequences relative to a random genomic library. Approximately 900,000 gene-enriched reads from maize were generated and clustered into Assembled Zea mays (AZM) sequences. Here we report the current AZM release, which consists of approximately 298 Mb representing 243,807 sequence assemblies and singletons. In order to provide a repository of publicly available maize genomic sequences, we have created the TIGR Maize Database (http://maize.tigr.org). In this resource, we have assembled and annotated the AZMs and used available sequenced markers to anchor AZMs to maize chromosomes. We have constructed a maize repeat database and generated draft sequence assemblies of 287 maize bacterial artificial chromosome (BAC) clone sequences, which we annotated along with 172 additional publicly available BAC clones. All sequences, assemblies and annotations are available at the project website via web interfaces and FTP downloads.

Approximately 80% of the maize genome comprises highly repetitive sequences interspersed with single-copy, gene-rich sequences, and standard genome sequencing strategies are not readily adaptable to this type of genome. Methodologies that enrich for genic sequences might more rapidly generate useful results from complex genomes. Equivalent numbers of clones from maize selected by techniques called methylation filtering and High C 0 t selection were sequenced to generate ∼200,000 reads (approximately 132 megabases), which were assembled into contigs. Combination of the two techniques resulted in a sixfold reduction in the effective genome size and a fourfold increase in the gene identification rate in comparison to a nonenriched library.

The readily scalable catching by polymerization purification technology has been further advanced to purify 5′-phosphorylated synthetic oligodeoxynucleotides (ODNs). The new technology utilizes a phosphoramidite that contains a fluoride-cleavable diisopropylsilyl acetal linker and a polymerizable methacrylamide group, and is capable of phosphorylation of ODN. For purification, the phosphoramidite was coupled to the 5′-end of full-length ODN on a synthesizer. Because failure sequences were capped in each synthetic cycle, only the full-length sequences were phosphinylated and acrylated. After cleavage and deprotection, the crude ODN was subjected to polymerization under typical acrylamide gel formation conditions. The full-length ODN was incorporated into polymer. The failure sequences and other impurities were simply removed by washing with water. Pure full-length ODN that contained a 5′-phosphate group was cleaved from the polymer with HF–pyridine. Reversed-phase (RP) HPLC showed that the ODN was pure, and the recovery yield was higher than that of typical preparative HPLC purification.