Salvia miltiorrhiza Bunge is an important traditional Chinese medicine (TCM). In this study, two S. miltiorrhiza genotypes (BH18 and ZH23) with different phenolic acid concentrations were used for de novo RNA sequencing (RNA-seq). A total of 170,787 transcripts and 56,216 unigenes were obtained. There were 670 differentially expressed genes (DEGs) identified between BH18 and ZH23, 250 of which were upregulated in ZH23, with genes involved in the phenylpropanoid biosynthesis pathway being the most upregulated genes. Nine genes involved in the lignin biosynthesis pathway were upregulated in BH18 and thus result in higher lignin content in BH18. However, expression profiles of most genes involved in the core common upstream phenylpropanoid biosynthesis pathway were higher in ZH23 than that in BH18. These results indicated that genes involved in the core common upstream phenylpropanoid biosynthesis pathway might play an important role in downstream secondary metabolism and demonstrated that lignin biosynthesis was a putative partially competing pathway with phenolic acid biosynthesis. The results of this study expanded our understanding of the regulation of phenolic acid biosynthesis in S. miltiorrhiza. Salvia miltiorrhiza Bunge is a widely used traditional Chinese medicine (TCM) with multiple clinical and pharmacological effects associated with cardiovascular, cerebrovascular, and hyperlipidemial diseases . H. H S. mihttps://doi.org/10.1155/2017/9364594). RA biosynthesis begins with the aromatic amino acids, L-phenylalanine and L-tyrosine, which are separately converted to the intermediate precursors 4-coumaroyl-CoA and 4-hydroxyphenyllactic acid through the tyrosine-derived pathway and the phenylpropanoid pathway, respectively. These two intermediate precursors are then covalently coupled by several biological reactions to generate RA  was set as the threshold for a significant differential expression level. The identified differentially expressed genes (DEGs) were used for the GO and KO enrichment analyses. The GO enrichment analyses were performed using GOseq , 670 genes  were identified as significant differentially expressed genes (DEGs) between these two lines, which comprised 250 upregulated genes (accounting for 37.32% of the significant DEGs) and 420 downregulated genes (62.68%) in ZH23, respectively  and “biosynthesis of secondary metabolites” , were the most significantly enriched pathways.To investigate the biochemical pathways of the DEGs, we mapped the DEGs in terms in the KEGG database and compared these results with the complete transcriptome background. The biosynthetic pathway leading to Sal B and RA is thought to entail both phenylpropanoid and tyrosine-derived pathways. In total, 11 DEG unigenes were related to phenylpropanoid biosynthesis or related pathways. Sm4CL2, EC 6.2.1.12),” which is involved in the core-phenylpropanoid pathway. Enzyme 4CL plays an important role in the general phenylpropanoid pathway by catalyzing a series of aromatic substrates to form their corresponding hydroxycinnamoyl-CoA esters, which are key precursors for the biosynthesis of numerous phenylpropanoid derivatives, including RA, flavonoids, and lignin [Among the 11 DEGs, one DEG, comp49431_c1, was upregulated 1.582-fold in ZH23 and was annotated as “4-coumarate:CoA ligase 2 , had an expression level in BH18 that was 2.17-fold higher than ZH23. HCT catalyzes the synthesis of the shikimate and quinate esters of p-coumaric acid , which cbidopsis . In addibidopsis . SmPAL, SmC4H, Sm4CL1, Sm4CL2, SmTAT, and SmHPPR, which are reported to be associated with RA biosynthesis, were validated by qRT-PCR. Ubiquitin was used as an internal reference. The transcription levels of these genes were low in both lines compared with the internal reference gene , SmF5H (comp51337_c0), SmREF1 (comp24721_c0), SmCAD1 (comp48837_c0), and SmPOD (comp36960_c0) in BH18 were all higher than that in ZH23, which is also consistent with RNA-seq results.We also used qRT-PCR to investigate the gene expression patterns of 5 DEGs significantly enriched with phenylpropanoid biosynthesis Figures . The exp S. miltiorrhiza vary greatly between different genotypes [ S. miltiorrhiza genotypes with different concentrations of bioactive components will promote a relatively accurate understanding of the molecular mechanisms of secondary metabolism in S. miltiorrhiza, especially for phenolic acids biosynthesis. However, several previous transcriptome investigations of S. miltiorrhiza only used one specific cultivar or a single genotype. In this study, two genotypes with different concentrations of bioactive components were selected to analyze phenolic acids biosynthesis using RNA-seq data.For species without reference genomes, RNA-seq can effectively determine accumulation levels of mRNA that can be associated with gene function, and this may potentially be an underlying cause of phenotypic variation. Previous studies have demonstrated the Sal B concentrations inenotypes , 7, 8. T S. miltiorrhiza using different RNA-seq methods. Yan et al. [ S. miltiorrhiza.In RNA-seq, the read lengths and the assembled total lengths are important factors for gene function analysis . In recen et al.  selectedn et al.  used then et al.  generaten et al.  reportedn et al.  reportedIn addition to the phenolic acid-branched pathway, phenylpropanoid metabolism also transforms phenylalanine into a variety of molecules, including lignins and flavonoids such as flavanones, dihydroflavonols, and anthocyanins. Thus, the phenolic acid, lignin, and flavonoid biosynthetic pathways share a common core phenylpropanoid pathway in their early steps. S. miltiorrhiza. Some previously reported genes in S. miltiorrhiza were generally identified in our RNA-seq data, and most of them showed higher expression in root of ZH23 than that in BH18. A central role gene, Sm4CL, in the core phenylpropanoid pathway, was upregulated 1.52-fold in ZH23. In addition, we detected 7 unigenes annotated as “4CL,” excluding comp49431_c1, which was annotated as Sm4CL2. Another unigene with relatively low expression levels was annotated as Sm4CL1 (comp50706_c0), with RPKMs of 5.89 in ZH23 and 2.49 in BH18. This difference in expression is consistent with previous reports that suggest that Sm4CL2 rather than Sm4CL1 might be responsible for Sal B biosynthesis in S. miltiorrhiza roots [ S. miltiorrhiza, and one had the highest level of expression, with RPKMs of 121.72 in BH18 and 146.65 in ZH23; thus, there was no difference between the two lines, and its length was the longest at 3,608 bp. This finding implies that maybe there are more than two functional 4CLs in the S. miltiorrhiza transcriptome.A total of 25 genes encoding up to 13 enzymes are annotated as being involved in RA biosynthesis inza roots . The rem Sm4CL2, and also most of genes in common core phenylpropanoid pathway in BH18, coumarate-CoA co-precursors might have relatively lower expression patterns, thus “reducing income” for the entire downstream pathway. We considered three mainly specific branch pathways: flavonoids, phenolic acids, and lignin. There were no significant DEGs among 28 background unigenes in the flavonoid pathways. To date, the detailed phenolic acid-branched pathway from RA to Sal B has not been characterized. However, the DEGs involved in the lignin pathway indicated it was probably a significantly enriched alternative pathway. Five key enzyme genes in the lignin pathway, for example, HCT, F5H, CAD, REF1, and POX, were upregulated in BH18, which maybe increase lignin biosynthesis to a high level, which can be further shown by histochemical properties and lignin concentrations in roots. We found that a greater number of vascular rays and cells per ray were stained in BH18 ( S. miltiorrhiza plants with downregulated cinnamoyl CoA reductase (SmCCR) exhibited dwarfing phenotypes, and both of the S and G lignin monomers were decreased by more than 60%. In contrast, the biosynthesis of phenolic acids, Danshensu, RA, and Sal B, was significantly increased [ Arabidopsis Production of Anthocyanin Pigment 1 transcription factor (AtPAP1) via the cosuppression of SmCCR and caffeic acid O-methyltransferase (SmCOMT).As a result of the downregulation of in BH18 , and thencreased . Zhang encreased  also repAlthough these data show that cooverexpression of lignin pathway genes, for example, HCT, CAD, F5H, ERF1, and POD, enhanced the lignin accumulation in BH18, the lower expression of genes in the core common upstream pathway will finally result in lower Sal B and RA contents in BH18 compared with ZH23. This result indicated that expression of genes involved in the core common upstream pathway plays a more important role in downstream second metabolism and also obviously indicated the lignin pathway as potential competing pathway with Sal B biosynthesis.Table S1: Primers used for gene expression analysis for qRT-PCR.Table S2: The content of salvianolic acid component in two S. miltiorrhiza lines.Figure S1: Schematic overview of the phenolic biosynthesis pathway in Salvia miltiorrhiza.Figure S2: Pathway assignment based on KEGG.Figure S3: Frequency distribution of BH18 and ZH23 by reads per kilobase per million (RPKM).


Manihot esculenta). In this study, 15 MeGRFs were identified from the cassava genome and were clustered into the ε and the non-ε groups according to phylogenetic, conserved motif, and gene structure analyses. Transcriptomic analyses showed eleven MeGRFs with constitutively high expression in stems, leaves, and storage roots of two cassava genotypes. Expression analyses revealed that the majority of GRFs showed transcriptional changes under cold, osmotic, salt, abscisic acid (ABA), and H2O2 treatments. Six MeGRFs were found to be commonly upregulated by abiotic stress, ABA, and H2O2 treatments, which may be the converging points of multiple signaling pathways. Interaction network analysis identified 18 possible interactors of MeGRFs. Taken together, this study elucidates the transcriptional control of MeGRFs in tissue development and the responses of abiotic stress and related signaling in cassava. Some constitutively expressed, tissue-specific, and abiotic stress-responsive candidate MeGRF genes were identified for the further genetic improvement of crops.GENERAL REGULATORY FACTOR (GRF) proteins play vital roles in the regulation of plant growth, development, and response to abiotic stress. However, little information is known for this gene family in cassava ( GENERAL REGULATORY FACTOR (GRF) proteins are also named 14–3–3 proteins that are widely distributed in eukaryotes. They are characterized as the most important phosphopeptide-binding proteins, playing a regulatory role in various biological processes ,3,4. ThrOryza sativa) [Arabidopsis thaliana [Glycine soja) [Gossypium hirsutum) [Populus trichocarpa [Brassica rapa [Brachypodium distachyon [Arabidopsis [G. soja [BdGF genes showed induction after abiotic stresses or hormone treatments in B. distachyon and overexpression of BdGF14a improved plant tolerance to drought stress [BdGF14d increased plant tolerance to salt stress by regulating ABA pathway, reactive oxygen species (ROS) scavenging system, and ions transports [Arabidopsis [In 1992, the first plant 14–3–3 protein was characterized from maize . Then, 1 sativa) , 13 fromthaliana ,12, 18 firsutum) , twelve irsutum) , 14 fromchocarpa , 21 fromstachyon . Furtherbidopsis . Multiplbidopsis ,23. GsGF[G. soja . Gh14–3–[G. soja . Additio[G. soja . AtGCN4 [G. soja . BdGF get stress . Overexpbidopsis . TogetheCassava is the sixth most important crop after wheat, rice, maize, potato, and barley . Its ediManihot esculenta ssp. flabellifolia), originally collected in Brazil, is the nearest ancestor of cultivated cassava, and has the characteristics of low photosynthesis rate, tuber root yield, and starch content in root tubers. Arg7 (M. esculenta Crantz cv. Arg7), adapted to geographical high-latitude region of Argentina, is a variety containing elite agronomic traits, including a certain level of growth under moderate drought stress. W14 and Arg7 were cultured in pots with a mixture of soil and vermiculite (1:1) under a growth room with a 16 h/35 °C day and 8 h/20 °C night regime, and a relative humidity of 70%. Stems (90 days after planting), leaves (90 days after planting), and storage roots (150 days after planting) in Arg7 and W14 were collected to study the transcriptional changes of cassava genes in different organs under normal growth conditions. For osmotic, cold, salt, ABA, and H2O2 treatments, Arg7 variety was cultured with Hoagland solution  under normal growth conditions. Sixty-days-old seedlings of Arg7 were subjected to aqueous solutions of 200 mM mannitol for 14 d, 300 mM NaCl for 14 d, 100 µM ABA for 24 h, 10% H2O2 for 24 h or low temperature (4 °C) for 48 h, respectively. Then, the leaves were sampled to perform quantitative real-time polymerase chain reaction (qRT-PCR) analysis.W14, (Arabidopsis and rice were acquired from UniProt and rice genome annotation project (RGAP), respectively [Arabidopsis as queries [Arabidopsis, and rice [The GRF protein sequences in ectively ,34. The ectively . The knoectively ,37. The  queries . Based o queries ,40. To sand rice ,42. The Total RNA of stems, leaves, and storage roots in Arg7 and W14 was extracted with plant RNA extraction kit  for complementary DNA (cDNA) library construction. The sequencing was performed with an Illumina GAII  following manufacturer’s instructions. Adapter sequences were removed with FASTX-toolkit . Clean rArabidopsis were constructed, which identifies 18 high confidence interactive proteins in Arabidopsis. Then, the homologs of these interactive proteins in cassava were identified with reciprocal best BLASTP analysis.Using the ExPASy proteomics server, the molecular weight (MW) and isoelectric points (pI) of cassava GRFs were predicted . The conGRFs were examined by qRT-PCR analysis using StratageneMx3000P  instrument and SYBR Premix Ex Taq . The 2−ΔΔCt method was used to assess the relative expression of cassava GRF genes [GRF genes in each time point was calculated according to the control and treated samples. Each sample has three replicates and three biological experiments were performed.The relative expression levels of cassava RF genes . The appArabidopsis and rice as query. As a result, 15 predicted full-length MeGRFs (MeGRF1–MeGRF15) were identified from cassava and their sequences were shown in Both BLAST and Hidden Markov Model searches were employed to extensively identify cassava GRF genes with GRF sequences from Arabidopsis  and cultivated variety Arg7  showed constitutively high expression (FPKM > 29) in all the tested tissues of the two genotypes. Interestingly, MeGRF2 showed tissue specific expression pattern, with transcripts lacking in leaves and low expression (FPKM < 10) in stems and storage roots of the two genotypes. MeGRF9 showed high expression in stems and storage roots of Arg7, but low expression in leaves of Arg7 and in all the tested tissues of W14. MeGRF14 had high expression in storage roots of Arg7, whereas low expression in other tissues tested. These results implied the same or differential roles of cassava GRF genes in tissue development.To study the expression profiles of ety Arg7 . The resMeGRF genes to abiotic stress and related signaling, cassava seedlings were subjected to cold, osmotic, salt, ABA, and H2O2 treatments , and rice (4–7). Conserved motif analysis showed that at least six 14–3–3 protein motifs existed in both ε group and non-ε group of cassava GRFs, indicating their conservation of protein sequences  in leaves, stems, and storage roots of W14 and Arg7 , MeGRF5 (homologs of AtGF4), and MeGRF-11, -12, -13 (homologs of AtGF6) in cassava also had high expression , suggesting their potential role in abiotic stress response (GRFs at the transcriptional level under abiotic stress were also observed in other plants. In banana, nine, 13, and twelve MaGRF genes showed induction after cold, salt, and osmotic treatments, respectively, whereas 10, six, and seven MaGRF genes showed repression under the corresponding treatments [B. distachyon, GRF genes could be transcriptionally induced or repressed after osmotic, salt, ABA, and H2O2 treatments [GRFs were upregulated after cold treatment, and were induced or repressed upon drought and salt treatments [B. rapa, most of the GRFs showed upregulation after salt, ABA, or SA treatments, but downregulation after dehydration or heat treatments [GRFs, further supporting the possible role of MeGRFs in abiotic stress responses.Biochemical and genetic evidences also confirmed the regulatory role of hormones ,29,54,55response . The sigeatments . In B. deatments . In commeatments . In mesoeatments . These eMeGRF3, MeGRF5, MeGRF6, and MeGRF11 were commonly upregulated by salt, osmotic, and ABA treatments; MeGRF4 was commonly upregulated by cold, salt, ABA, and H2O2 treatments; and MeGRF10 was commonly upregulated by cold, osmotic, and H2O2 treatments (GRFs in plants response to abiotic stress through affecting ABA pathway, stomatal behavior, ROS balance, and ions transports [GRFs may be the converging points when cassava responds to abiotic stress, ABA signaling and H2O2, and can serve as candidates for genetic improvement of crop tolerance to abiotic stress.Notably, eatments . Numerouansports ,27,28,29Arabidopsis [As an important regulatory factor, GRFs elaborate their function through interacting with different clients. There is a need to investigate GRF mediated interaction network. In this study, we predicted 18 possible targets of 13 GRFs in cassava, including enzymes, transporters, protein kinases, and transcription factors . These ibidopsis ,4,5,6,7.MeGRFs. Expression analysis revealed the involvement of MeGRFs under abiotic stress and related signaling and identified some important candidates for improving crop resistances to multiple stresses. Furthermore, the GRF mediated interaction network was characterized, which would facilitate further study of their function in cassava. This systematic study will advance the understanding of GRF-mediated signal cascades in regulating cassava development and abiotic stress response, thereby supplying candidates for crop breeding.In conclusion, this study identified 15 GRFs from cassava and investigated their phylogenetic classification, protein motif, and gene structure. Transcriptomic analysis showed the constitutively expressed or tissue specifically expressed


Next to T cell receptor and cytokine signals, membrane-bound Notch ligands have an important role in orchestrating Th cell differentiation. Several studies provided evidence that DC activation is accompanied by surface expression of Notch ligands. Intriguingly, DCs that express the delta-like or Jagged Notch ligands gain the capacity to instruct Th1 or Th2 cell polarization, respectively. However, in contrast to this model it has also been hypothesized that Notch signaling acts as a general amplifier of Th cell responses rather than an instructive director of specific T cell fates. In this alternative model, Notch enhances proliferation, cytokine production, and anti-apoptotic signals or promotes co-stimulatory signals in T cells. An instructive role for Notch ligand expressing DCs in the induction of Th cell differentiation is further challenged by evidence for the involvement of Notch signaling in differentiation of Th9, Th17, regulatory T cells, and follicular Th cells. In this review, we will discuss the two opposing models, referred to as the “instructive” and the “unbiased amplifier” model. We highlight both the function of different Notch receptors on CD4+ T cells and the impact of Notch ligands on antigen-presenting cells.For protection against pathogens, it is essential that naïve CD4 Since the original observation in 1986 upon antigenic stimulation naive CD4+ T cells can differentiate into T helper 1 (Th1) or Th2 effector T cells depending on polarizing cytokine signals  and the micro-environment, activated CD4 signals , 4.Tbx21 , encoded by Tbx21 . Th2 celTbx21 . BecauseTbx21 , 8.An accumulating number of studies suggest that the Notch signaling pathway, which also plays a crucial role in early hematopoietic development and at multiple steps of T lineage development, is essential for Th cell differentiation [for recent review see Ref. ]. CurrenThere are five Notch ligands: two Jagged (Jagged1 and Jagged2) and three DLL , which are bound by four receptors, Notch1–4. For these ligands to be functional, their ubiquitination by Mindbomb1 or Neuralized within the cell is required . DetailsT helper 2-promoting stimuli including helminth eggs, prostaglandin E2, cholera toxin, and allergens, such as house dust mite (HDM), birch pollen, and cockroach allergens, were shown to induce Jagged expression on APCs, as summarized in Table Il4 gene and (ii) an upstream promoter of Gata3  a 3′ enhancer of the of Gata3 , 53–55. of Gata3 , 56. Wheof Gata3 –59. Moreof Gata3 . At the en Table  and in aen Table . A prote in vivo .Trichuris muris in DN-MAML transgenic mice were restored when mice received anti-IFN-γ antibodies, indicating that Notch functions to optimize rather than to initiate the Th2 response (in vivo HDM-exposure (However, defective Th2 responses against the intestinal helminth response . Moreoveresponse . Finallyexposure . Althougexposure , our stuTaken together, although several lines of evidence indicate that DCs use the Notch pathway to instruct Th cell fates, Notch may also act as an unbiased amplifier of Th cell differentiation.Ifng and Tbx21 were identified as direct Notch targets (in vivo Leishmania major parasite infection, but reports employing DN-MAML transgenic or conditional RBPJκ knockout mice demonstrated that Th1 cell function was unaffected (in vivo EAE model (The signature Th1 genes  targets , 61. Micaffected , 56, 62.AE model , 61. By AE model . The intAE model .in vitro and in vivo experiments, as outlined in Table in vivo (in vitro (The capacity of DLL1/DLL4 to induce Th1 cell differentiation is supported by many  in vivo , 49, 50. in vivo . Convers in vivo , 48. Genin vitro , 30.In conclusion, although most studies would support an instructive role for DLL1/DLL4 in Th1 induction, the role of Notch signaling in Th1 cell differentiation remains incompletely understood.via Jagged2 ligation (Rorc, Il17, and Il23r gene promoters are direct Notch targets and, accordingly, Th17 cell differentiation is impaired when Notch signaling is blocked (in vitro (in vivo autoimmune models (via Jagged1 and DLL4, respectively (Given the increasing complexity of T cell subset biology, it is not unexpected that the bipotential instructional model is not sufficient to fully explain the function of Notch signaling in Th cell differentiation. For example, Notch signaling cooperates with TGF-β to induce Th9 cell differentiation and IL-9 expression ligation . The Ror blocked –70. Here blocked , 60, 71, blocked –74. Remain vitro , althougin vitro . Differein vitro –80, wherin vitro –88. Althin vitro , the role models , 90, 91.ectively , 92, 93.ectively , 95, impvia upregulation of DLL or Jagged ligands on DCs (Figure As summarized in Table s Figure . This ims Figure , the molN-acetylglucosamine preferentially signal via delta ligands, while Jagged binding is inhibited (It has been shown that DLL4 induces a stronger Notch signal than DLL1 or Jagged1 . Also, tnhibited . Absencenhibited , 98. Anonhibited , 53. Thenhibited , 99, 100nhibited , distincnhibited . Howevernhibited  and thatnhibited , 61. Thinhibited . It is, in vitro in the absence of polarizing cytokines (via DLL4 was shown to boost antigen sensitivity of CD4+ T cells via promoting co-stimulatory signals in T cells (+ T cells overcome a Th cell commitment threshold. In support of a role for Notch as an unbiased amplifier (Figure in vitro of both Th1 and Th2 cells (Several studies are in apparent conflict with the “instructive model.” For example, DLL were reported to promote Th2 responses or Jagged ligands were implicated in Th1 induction , 104. Neytokines . Importaytokines . In addi T cells . Togetheh2 cells , 18, 105h2 cells , 106.in vitro HDM-pulsed bmDCs were used for allergen sensitization, but not when mice were in vivo sensitized by endogenous airway DCs (via CD46 and CD3 was shown to up regulate Jagged1 on human T cells (in vivo.It is conceivable that minor differences in experimental design or conditions form the basis of the discrepant results that support one of the two opposing models for Notch function in Th differentiation. Many studies on Notch ligands on APCs have employed GM-CSF cultured bmDCs Table , which wrway DCs . Moreoverway DCs , 99, 108 T cells , suggest T cells , which w T cells . Indeed, T cells , 18, 22. T cells . More exin vivo Th2 cell differentiation may point at Tfh rather than Th2 defects and should, therefore, be interpreted with care.Another concern is that some gain-of-function approaches, involving overexpression of Notch receptors or ligands, may be associated with strong or prolonged, less physiological Notch signals. In this context, it is interesting that variable Notch signal strength allows induction of distinct responses by the same signaling pathway , 113, pain vivo studies, using cell-specific knockout of individual Notch ligands or combinations.Given the increasing number of characterized Th subsets, it is unlikely that Notch signaling simply acts as a bimodal molecular switch for the induction of either Th1 and Th2 differentiation, based on DLL and Jagged expression on DCs, respectively. Nevertheless, many studies described above support the notion that individual Notch ligands have differential effects on Th cell differentiation, which cannot be explained by the unbiased amplifier model. The two models, however, may not necessarily be mutually exclusive. Effects of Notch signaling could be quite different during induction and during maintenance of Th subset differentiation. Moreover, the finding that there is quite some plasticity between Th subsets (Since Notch signaling is involved in the differentiation of basically all Th subsets, it could serve as a potential therapeutic target, for example, by inhibiting Th2 responses in allergies or Th1/Th17 responses in autoimmune diseases. However, because effects of GSI are not limited to Notch signaling, it will be valuable to develop more specific compounds targeting Notch signaling components. Indeed synthetic, cell-permeable stabilized peptides that target a critical protein–protein interface in the Notch transactivation complex –120 as wInterestingly, GSI administration during only the challenge in asthma models was sufficient to decrease Th2 cytokine production , 59. TheIT and MP performed the literature research, wrote the paper, and designed the figure. RH critically revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The present study was conducted to assess the gender difference in the relationship between serum ferritin and 25-hydroxyvitamin D [25(OH)D] in Korean adults.A total of 5,147 adults  aged ≥ 20 years from the Korean National Health and Nutrition Examination Survey (KNHANES) data (2012) were analyzed. A covariance test adjusted for covariates was performed for serum ferritin levels in relation to vitamin D status D < 10.0 ng/mL; vitamin D insufficiency, 25(OH)D ≥ 10.0, < 20.0 ng/mL; vitamin D sufficiency, 25(OH)D ≥ 20.0 ng/mL).P = 0.012). Second, in premenopausal women, after adjusting for related variables, serum ferritin levels were increased with the increasing of serum 25(OH)D level (P = 0.003). Third, in postmenopausal women, after adjusting for related variables, serum ferritin levels were not significantly increased with the increasing of serum 25(OH)D level (P = 0.456).The key study results were as follows: First, in men, in terms of serum ferritin levels by serum 25(OH)D level after adjusting for age, smoking, alcohol drinking, regular exercise, SBP, DBP, WM. TC, TGs, HDL-C, FPG, Hb, Hct, MCV, and Fe, serum ferritin levels were inversely increased with the increasing of serum 25(OH)D level (Serum 25(OH)D level was inversely associated with the serum ferritin levels in men, but was positively associated with the serum ferritin levels in premenopausal women, and was not associated with the serum ferritin levels in postmenopausal women. Vitamin D is involved in calcium and phosphate absorption in the intestines, whereby it maintains sufficient concentrations of circulating calcium and phosphate levels as well as normal mineralization of bone by providing the minerals to bone-forming sites . RecentlIron is a ubiquitous metal of vital importance to the normal physiologic processes of many organism  and it iResearch on the association between vitamin D and ferritin is rare. In addition, the association between vitamin D and ferritin is still being debated because the findings vary across studies. One study reported that vitamin D is positively associated with ferritin . Howeverhttp://knhanes.cdc.go.kr) is currently operating an English-language information homepage. The data of the respective year are available to everyone at the free of charge. If the applicant enters simple subscription process and his/her email address in the official website of KNHANES, the data of the respective year can download to free of charge. If additional information is required, the readers can contact the department responsible for data .KNHANES V-3 data were collected for 1 year (2012), using a rolling sampling survey that involved a complex, stratified, multistage, probability cluster survey of a representative sample of the non-institutionalized civilian population in South Korea. The survey was composed of three parts: a health interview survey, a health examination survey, and a nutrition survey. Each survey was conducted by specially trained interviewers. The interviewers were not provided with any prior information regarding specific participants before conducting the interviews. Participants provided written informed consent to participate in this survey, and we received the data in anonymized form. In the KNHANES V-3 (2012), 8,058 individuals over age 1 were sampled for the survey. Among them, of the 6,221 subjects who participated in the KNHANES V-3, we limited the analyses to adults aged ≥ 20 years. We excluded 874 subjects whose data were missing for important analytic variables such as serum ferritin level, 25(OH)D level, various blood chemistry tests, and information about lifestyle. In addition, we excluded participants who had liver cancer (57 subjects), hepatitis virus B (126 subjects), and hepatitis virus C (17 subjects). Finally, 5,147 subjects  were included in the statistical analysis. The KNHANES V-3 study has been conducted according to the principles expressed in the Declaration of Helsinki. . All participants in the survey signed an informed written consent form. Further information can be found in “The KNHANES V-3 (2012) Sample”, which is available on the KNHANES website. The official website of KNHANES , smoking (non-smoker or ex-smoker or current smoker), alcohol drinking (yes or no), and regular exercise (yes or no). In the smoking category, participants who smoked more than one cigarette a day, those who had previously smoked but do not presently smoke, and those who never smoked were classified into the current smoker, ex-smoker, and non-smoker groups, respectively. Alcohol drinking was indicated as “yes” for participants who had consumed at least one glass of alcohol every month over the last year. Regular exercise was indicated as “yes” for participants who had exercised on a regular basis regardless of indoor or outdoor exercise. . Anthropometric measurements included measurement of body mass index (BMI) and waist measurement (WM), as well as final measurements of systolic blood pressure (SBP) and diastolic blood pressure (DBP). Blood chemistries included measurements of total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), triglycerides (TGs), fasting plasma glucose (FPG), 25-hydroxyvitamin D [25(OH)D], serum iron (Fe), total iron binding capacity (TIBC), transferrin saturation (TFS), hemoglobin (Hb), hematocrit (Hct), and mean corpuscular volume (MCV).125 I RIA Kit; DiaSorin, Still Water, MN, USA) using a 1470 Wizard Gamma Counter . To minimize the analytical variation, serum 25(OH)D levels were analyzed by the same institute, which carried out a quality assurance program through the analysis period. Serum 25(OH)D levels were classified as either vitamin D deficiency [25(OH)D < 10.0 ng/mL], vitamin D insufficiency , or vitamin D sufficiency [25(OH)D ≥ 20.0 ng/mL]  for the measurement of vitamin status and we also used the 25(OH)D3 level in the blood. However, recent studies uncovered novel metabolic pathways in vitamin D metabolism in human serum and tissues. These studies suggested that vitamin D3 acts as a substrate for cytochrome P450scc (CYP11A1), and CYP11A1 hydroxylases D3 to produce 20-hydroxyvitamin D3 and 20,22 dihydroxyvitamin D3  . Most sttamin D3 –43. In a 25(OH)D ,43. CYP1 25(OH)D ,45 and i 25(OH)D . TherefoVitamin D plays a role in preventing anemia but also decreases oxidative stress, insulin resistance, and inflammation ,24,47. FThe present study has some limitations. First, hepcidin is an important determinant of ferritin. However, hepcidin were not employed in the KNHANES V-3 study (2012). Second, serum calcium concentrations and the daily intake volume of vitamin D are important determinants of serum 25(OH)D levels, but the KNHANES V-3 did not measure serum calcium concentrations or daily intake volumes of vitamin D. Therefore, serum calcium concentrations and daily intake volumes of vitamin D could not be used as adjustment variables. Third, sexual hormones, such as estrogens and testosterone, are an important determinant of serum 25(OH)D and ferritin. However, estrogens and testosterone were not employed in the KNHANES V-3 study (2012). The serum 25 (OH)D for each season, along with calcium, hepcidin, estrogens, and testosterone levels, should be included as variables for serum 25(OH)D in future studies. Although the present study has these limitations, this is the first reported study to determine gender differences in the relationship between ferritin and vitamin D in Korean adults. Therefore, more accurate results might be obtained by performing a cohort study by adding these variables.The present study investigated the association between serum ferritin levels and vitamin D using data from the fifth KNHANES conducted in 2012. Serum 25(OH)D level was inversely associated with serum ferritin levels in men. Conversely, serum 25(OH)D level was positively associated with serum ferritin levels in premenopausal women and was not associated with serum ferritin levels in postmenopausal women.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Fetal alcohol spectrum disorder (FASD) is associated with long-termdeficits in cognitive and motor functions. Previous studies linkedneurodevelopmental abnormalities to increased oxidative stress and whitematter hypotrophy. However, similar effects occur with low-dose nitrosamineexposures, alcohol abuse correlates with cigarette smoking, and tobaccosmoke contains tobacco-specific nitrosamines, including NNK.Tobacco smoke exposure is a co-factor in FASD.Long Evans rat pups were i.p. administered ethanol (2 g/kg) onpostnatal days (P) 2, 4, 6 and/or NNK (2 mg/kg) on P3, P5, and P7 tosimulate third trimester human exposures. Oligodendroglialmyelin-associated, neuroglial, and relevant transcription factor mRNAtranscripts were measured using targeted PCR arrays.Ethanol and NNK differentially altered the expression of immature andmature oligodendroglial, neuronal and astrocytic structural andplasticity-associated, and various transcription factor genes. NNK’seffects were broader and more pronounced than ethanol’s, andadditive or synergistic effects of dual exposures impacted expression of allfour categories of genes investigated.Developmental exposures to alcohol and NNK (via tobacco smoke)contribute to sustained abnormalities in brain white matter structure andfunction via distinct but overlapping alterations in the expression of genesthat regulate oligodendrocyte survival, maturation and function, neuroglialstructural integrity, and synaptic plasticity. The results support thehypothesis that smoking may contribute to brain abnormalities associatedwith FASD. The cerebellum, temporal lobe, and white matter aremajor targets of ethanol’s teratogenic effects, and while there is goodevidence that ethanol-induced pathology in the cerebellum and temporal lobe islinked to impaired insulin and insulin-like growth factor type 1 (IGF-1) signalingthrough Akt-mediated growth, cell survival, metabolic, and neuronal plasticitynetworks, the nature and pathogenesis of white matter hypotrophy are poorlyunderstood. However, since growth factor, including IGF-1 signaling throughphosphatidylinositol-3-kinase (PI3K) and Akt promotes oligodendrocyte survival andfunction, including myelin synthesis, maintenance, andhomeostasis–5,impairments in insulin/IGF signaling that occur with alcohol exposure couldpotentially underlie the white matter abnormalities in FASD. Since the white matterhypotrophy may be due to reduced myelin synthesis, maturation and, maintenance orprimary loss of axons, evaluating the state of oligodendrocyte function could aidour understanding of its molecular pathogenesis.Heavy chronic or binge alcohol exposures during development lead to sustaineddeficits in cognitive and motor functions. The characteristic constellation ofcraniofacial abnormalities together with brain structural pathology is referred toas fetal alcohol spectrum disorder (FASD)–8. Finally, mature myelinating oligodendroglia furtherexpress distinct integral membrane proteins including, myelin basic protein (MBP),myelin associated glycoprotein (MAG), myelin oligodendrocyte glycoprotein (MOG), andproteolipid protein (PLP),4,9. Oligodendroglial differentiation is regulated byWnt/β-catenin  via the Tcf4 transcriptionfactor, Notch1-HES5, and insulin/IGF signaling–19.Oligodendroglia produces central nervous system (CNS) myelin. Myelin has anabundant lipid composition (70 – 85%) with lower proportions ofproteins (15 – 30%) and water compared with other cellularmembranes. High lipid content ensures optimum nerve cell conduction.Oligodendrocytes develop from precursor cells (OPC 1–3) that differentiallyexpress Cnp, Ng, PDGF-Rα, Olig2, Dix2, and Nkx. OPCs differentiate intoimmature oligodendroglia that express Cnp, Olig1, and low levels of Olig2, whichmature and express Cnp, Olig1, low Olig2, and RTN4and MAG, andde novo myelin synthesis. White matter integrity is crucial for CNS functionsince reduced brain myelination during development results in cognitiveimpairment. Mixed glial cultures were used to demonstrateoligodendrocyte’s extreme vulnerability to the toxic effects of ethanolcompared with other cell types. The pro-death effects of ethanol are mediated byinhibition of PI3K and Erk MAPK. Ethanol impaired signaling through PI3K/Akt isdue to reductions in insulin/IGF receptor binding[ and fluidity , as well as decreasedphosphorylation and activation of downstream pathways–31.Chronic ethanol exposure delays oligodendroglial maturation, expression ofMBPr binding associat. Review of an outpatient substance abuse treatmentcenter database for pregnant women revealed that from 2010 to 2013, 74% ofthe pregnant alcohol users (N = 57) smoked cigarettes compared with42% of controls (N = 31) (P = 0.0053). Despite strong evidencelinking cigarette smoking during pregnancy to impaired fetal growth and development,and neurocognitive function, the mechanisms are not well understood. Given thefrequency of overlapping exposures and their known independent adverse effects ondevelopment, further research is needed to determine the degree to which alcohol andtobacco exposures produce differential or additive adverse effects on white matterin the immature brain.Despite evidence that ethanol exposures can be sufficient to cause FASD,variability in the nature and severity of the phenotype suggests that co-factors maycontribute to its pathogenesis. Of note is that a very high percentage of heavydrinkers (up to 80%) also abuse tobacco products, mainly by smokingcigarettes–38,particularly in relation to the tobacco-specific nitrosamine,4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and itsmetabolites, it is known that low, sub-mutagenic exposures tonitrosamines such as streptozotocin, N-nitrosodimethylamine (NDEA), and NNK causeinsulin resistance with impaired signaling through PI3K-Akt, increased DNA damage,lipid peroxidation, mitochondrial dysfunction, and ER stress–42. The present study tests the hypothesis thatsub-mutagenic exposures to NNK are sufficient to cause white matter molecularpathology similar to the effects of ethanol, and possibly exacerbate the adverseeffects of ethanol with respect to impairments in oligodendrocyte and neuroglialgene expression.Although the vast majority of research designed to investigatealcohol-tobacco interactive effects has been focused oncarcinogenesis–45, and NNK was administeredon P3, P5, P7, and P9. These models simulated 3rd trimester-equivalent humanpregnancy exposures to alcohol and tobacco toxins. The rats were sacrificed at 6weeks of age to examine long-term effects on temporal lobe insulin/IGF-1 signalingthrough Akt growth and metabolic pathways during late adolescence. All experimentswere performed in accordance with protocols approved by Institutional Animal Careand Use Committee at the Lifespan-Rhode Island Hospital, and they conformed toguidelines established by the National Institutes of Health.Long Evans rat pups were divided into 4 groups and administered 50μl i.p. injections of: saline vehicle as control; pharmaceutical gradeethanol ; NNK ; and ethanol + NNK.Ethanol treatments (binge) were administered on postnatal days (P) 2, 4, 6, and8“). The objective was to evaluate ethanol and NNK effects ongenes relevant to CNS myelin and white matter development and integrity. PCR primerpairs were designed with MacVector 12  and Primer 3 (http://primer3.sourceforge.net/) software (“). Targeted arrays were constructed by spotting and dryingprimer pairs (100 nmol/5 μl) into individual wells of a Light cycler 480Multi-well Plate 96 . The arrays were sealed and stored at−80°C. On the day of use, the array plates were thawed on ice and 20μl of reaction cocktail containing cDNA from 5 μg RNA template andSybr green master mix were added to each well. PCR reactions were performed in aRoche Lightcycler 480 System. Gene expression was analyzed using theΔΔCt method with results normalized to housekeeping control genes.Targeted quantitative RT-PCR arrays were used to measure expression of geneslinked to oligodendrocyte and astrocyte function  with Tukey orlinear trend post hoc tests . F-ratios and P-valuesare tabulated. Significant post-test differences and trends (0.05 < P < 0.10)are shown in the graphs. Heat maps were constructed using Version 3.1 of R software.Exploratory analysis identified missing values verified the quality of observeddata. To scale the data, row means were subtracted from each cell. The resultingvalues were further divided by the standard deviation in order to obtain a z-scoreof each individual cell. Row values are scaled to have a mean = 0 andstandard deviation (S.D.) = 1. The resulting values were plotted using acosmetically modified heatmap library function in R and a 6-color palette. We alsoapplied hierarchical clustering algorithm using Euclidean distance function on theoverall table to display a dendrogram of mRNAs.PCR arrays were generated to measure expression of 84 genes involved inglial and neuronal growth, maturation, and function. Pilot screening enabled usto eliminate 55 genes due to their low levels of expression or absentinter-group differences. The follow-up studies were focused on 29 genes  in whichAs markers of immature oligodendroglia, we measured nestin, vimentin,PROM1, CNP, PDGFR-α, GC, and GalC, and for mature myelinatingoligodendroglia, we measured PLP, MOG, MAG-1, MBP, RTN4, RPAIN, and ST8sia1expression (Table 1). Significant ethanol effects were observed with respect toPROM1, GC, GalC, MAG-1, RPAIN, and ST8sia1, and trend effects were observed fornestin and PLP. Significant NNK effects were detected for vimentin, PROM1,PDGFR-α, GC, GalC, PLP, MOG, MBP, RTN4, RPAIN, and ST8SIa1, and trendeffects occurred for CNP and MAG-1 expression. Significant ethanol x NNKinteractive effects occurred for PROM1, GalC, PLP, MBP, and RPAIN, and trendeffects were observed for GC.Graphed results and post hoc repeated measures tests revealedsignificant intergroup differences with respect to all immature and maturemyelin-associated genes except for CNP . EthanolNNK and particularly ethanol + NNK had broader effects on theexpression of both immature and mature oligodendroglial genes, and in severalinstances, combined exposures produced additive or synergistic responses. NNKsignificantly increased GalC ,PROM1 , PLP Fi, MOG Fi RTN4 Fi, and RPAHeatmaps corresponding to immature and mature oligodendroglial geneexpression are depicted in With regard to mature oligodendroglial genes, ethanol- and NNK-mediatedpatterns of altered gene expression were more consistent. MBP expression washighest in control brains, and prominently reduced by all other treatments.Otherwise, the expression levels of most genes increased with ethanol, NNK, andethanol + NNK treatments. For PLP, MOG, RTN4, RPAIN, and ST-8SIA1, geneexpression increased from ethanol to NNK. Except for MBP, matureoligodendroglial gene expression was highest in the ethanol + NNK group or similarly high in the NNK and ethanol + NNKgroups .The neural-glial mRNA transcripts measured included: CSPG4, GFAP, NCAM,NTRK2, GSTP1, GPD1 and GPD2. Two-way ANOVA tests demonstrated significanteffects of ethanol on GFAP, NCAM, and GPD2, and trend effects on NTRK2 (Table2). NNK had significant effects on GFAP, NTRK2, GSTP1, and GPD2, and trendeffects on CSPG4. Ethanol x NNK interactive effects were significant for NTRK2and GPD2, and had a trend effect on GFAP. Post hoc tests revealed that ethanol,GFAP  and NTRKGlial-associated transcription factor mRNA transcripts measuredincluded: FOXO1, FOXO4, NKX2-2, NKX6-1, Olig1, Olig2, PAX6, and SOX9. Two-wayANOVA tests demonstrated significant ethanol and ethanol x NNK interactiveeffects on FOXO1, FOXO4, NKX6-1, and PAX6, and NNK effects on all genes exceptOlig2 (Table 2). Post hoc tests revealed that ethanol significantly increasedexpression of NKX6-1  anddecrHeatmap analysis demonstrated that control temporal lobes had high mRNAlevels of GFAP, NTRK, and GPD1, intermediate levels of CSPG4, and low levels ofNCAM, GSTP1, and GPD2 . EthanolVery low levels of FOXO1, FOXO4, NKX2- 2, NK6-1, Olig1 and Olig2, andhigh levels of PAX6 and SOX9 in control temporal lobes, and sharply oppositetrends in the ethanol + NNK group . EthanolNNK’s effects were intermediate between those of ethanol andethanol + NNK , similar to those ofethanol + NNK , similar to control (FOXO1), or differentfrom the other groups (PAX6).; 2) the causes of progressive alcoholic braindisease are more complex than alcohol consumption per se; 3) previous studies showedthat exposures to sub-mutagenic levels of other nitrosamines causeneurodegeneration–41,46; and 4) we recently showed thatearly developmental exposures to ethanol and dietary nitrosamines used in preservedand processed foods, i.e. N-nitrosodiethylamine, differentially or additivelycontributed to cerebellar abnormalities associated with fetal alcohol spectrumdisorder. Our working hypothesis herein was that chronic lowdose NNK exposures mediate CNS degenerative abnormalities that overlap with andexacerbate adverse effects of alcohol. NNK rather than tobacco smoke effects werestudied because smoking could confound the results by causing pulmonarydisease,49.Ethanol and Tobacco Nitrosamine (NNK) Related Brain Disease: This study wasdesigned to examine the independent and interactive effects of ethanol and NNKexposures on neurodegeneration in adult rats. The chronic-binge ethanol exposuremodel was used to mimic the human clinical scenario that is most prevalent amongalcoholics. Chronic low-dose NNK tobacco-specific nitrosamine exposure effects wereof interest in relation to alcoholic neurodegeneration because: 1) a very highpercentage of heavy drinkers also smoke,51, illustrating howNNK’s effects can mimic those of ethanol.The results show that ethanol had limited effects on the expression ofimmature oligodendroglial genes and only increased GalC, whereas NNK, with orwithout ethanol co-exposures significantly modulated expression of 6 of the 7genes examined. Stimulatory effects occurred for GC, PROM1 and GalC, whileinhibitory effects occurred with respect to nestin, Vimentin, andPDGFR-α. Nestin regulates vimentin intermediate filament disassemblyduring growth and is needed for survival, renewal and proliferation of neuralprogenitor cells. Inhibition of Nestin corresponds with prior reports ofimpaired neuronal genesis in chronic ethanol-exposed experimentalanimals,53. The significantly elevated levels in ethanol+ NNK exposed temporal lobes suggest that the dual treatments may leadto accelerated maturation of oligodendroglia. At the same time, the increasedexpression of PROM1, particularly in the ethanol + NNK group could haveled to suppressed maturation of progenitor or stem cells,55, reducing the pool of myelin-generatingprecursors. GalC  hydrolyzes thegalactose ester bond of galactosylceramide in its lysosomal catabolism, andeither over-expression or deficiency can adversely affectoligodendrocyte maturation and function.PDGFR-β is a cell surface receptor tyrosine kinase expressed inoligoprogenitor cells and when stimulated with PDGF, activates proliferationpathways. Therefore, NNK inhibition would have impaired growth of immatureoligodendroglia. GC is a Vitamin D binding nuclear hormone receptor thatinteracts with RXR and regulates maturation of immature oligodendroglialprecursors into myelin-generating oligodendrocytesGene expression and heatmap analyses of mature oligodendroglial geneexpression profiles revealed progressive up-regulation of PLP1, MOG, MAG-1,RTN4, RPAIN, and ST8SIA1 from control, to ethanol, and then NNK exposures, andeither further increases with dual exposures, or similar maximum levels ofexpression as observed in either ethanol or NNK-exposed brains. The generallyhigher levels of both immature and mature myelin gene expression in ethanol+ NNK exposed brains relative to the other groups most likely reflectsresponses to injury in the early understand how these responses impact whitematter structure and function in adolescence. MBP was the exception to thesetrends in that the highest expression levels were in control brains. Ethanol andNNK conspicuously down-regulated MBP relative to control. Since MBP is a majorprotein constituent of myelin sheaths and has an important role in forming andstabilizing myelin membranes, irrespective of the up-regulation of other myelingenes, MBP’s down regulation would impair the integrity of mature whitematter myelin., and oligodendrocyte responses to stress (GPD1).NNK’s stimulatory, and ethanol + NNK’s furtherup-regulation of CSPG4 suggests that NNK exposures during development inhibitneurite outgrowth and promote growth cone collapse during axonregeneration,60 inadolescent brains, and that these effects are exacerbated by dual exposures toethanol and NNK. These findings correspond with NNK’s inhibition ofNCAM1expression, which mediates neuronal adhesion and neuriteoutgrowth,61. It is alsonoteworthy that the ethanol-only effects were opposite those of NNK with respectto these genes, as well as GSTP1 (up-regulated by NNK and not ethanol) whichnegatively regulates p25-mediated neurodegeneration, highlighting the divergenteffects of these agent exposures on subsequent CNS molecular pathology. Theoverall findings with respect to NNK’s effects on CSPG4, NCAM1, andNTRK2 suggest that NNK exposures via tobacco smoke late in development haslong-lasting deleterious effects on neuronal plasticity and repair in theadolescent brain.The collective responses of neuroglial genes to ethanol and/or NNKexposures were more variably patterned than those observed for oligodendrocyteand transcription factor genes. The reductions in GFAP, NTRK, and GPD1 withethanol and/or NNK exposures relative to control potentially carry significancewith respect to impairments in blood-brain barrier integrity (GFAP), synapticplasticity (NTRK)–65, and NKX2-2 controls genes thathave important roles in axonal guidance, the findings suggest that theobserved modulations in transcription factor gene expression may representcompensatory efforts to attenuate effects of impaired neuronal growth andplasticity, oxidative stress, and insulin resistance,65. However, the inhibitory effects on PAX6 andSOX9 suggest that ethanol and NNK impair mechanisms of glial progenitor cellmaintenance and eventual maturation–70, and thereby contribute to white matter pathologyin “FASD” that is mediated by developmental exposures to alcoholand/or tobacco smoke (NNK).The main findings with respect to transcription factors were that theexpression levels of FOXO1, FOXO4, NKKX2-2 NKX6-1, Olig1, and Olig2 wereincreased by ethanol and/or NNK exposures, and for the most part, furthersharply increased by ethanol + NNK exposures, reflecting additive orsynergistic effects. Opposite effects occurred with respect to PAX6 and SOX9.Since both FOXO1 and FOXO4 target insulin signallingMain effects of ethanol and NNK revealed by hierarchical clusteredanalysis of gene expression. Hierarchical clustering by heatmap analysisrevealed broad independent and additive or synergistic effects of ethanol andNNK exposures on temporal lobe gene expression . Two dom


Human adenovirus 7 (HAdV-7) strains are a major cause of acute respiratory disease (ARD) among adults and children, associated with fatal pneumonia. An ARD outbreak caused by HAdV-7 that involved 739 college students was reported in this article. To better understand the underlying cause of this large-scale epidemic, virus strains were isolated from infected patients and sequence variations of the whole genome sequence were detected. Evolutionary trees and alignment results indicated that the major capsid protein genes hexon and fibre were strongly conserved among serotype 7 strains in China at that time. Instead, the HAdV-7 strains presented three thymine deletions in the virus associated RNA (VA RNA) II terminal region. We also found that the mutation might lead to increased mRNA expression of an adjacent gene, L1 52/55K, and thus promoted faster growth. These findings suggest that sequence variation of VA RNA II gene was a potential cause of such a severe HAdV-7 infection and this gene should be a new-emerging factor to be monitored for better understanding of HAdV-7 infection. Human adenoviruses (HAdVs) are pathogenic viruses that can usually cause acute respiratory disease (ARD), acute follicular conjunctivitis, haemorrhagic cystitis, gastroenteritis, myocarditis, meningoencephalitis, and even death . Outbreavs. epidemic, or mild vs. severe [The severity of HAdV-7 infection depends on many factors, such as virus virulence, viral transmission environment, or physical condition of the host, and dictates whether occurrence is sporadic . severe –10. Gene. severe , 12. A bTraditionally, capsid hexon and fibre genes have been used to detect genomic variation, as they also determine the HAdV serotypes. However, according to recent reports, the hexon and fibre genes of HAdV-7 were remarkably conserved across time and space . With thHere, we report a large-scale outbreak of severe acute respiratory disease associated with HAdV-7 that occurred in Wuhan, China, in 2015. To assess whether this outbreak was related to genetic variation of the VA RNA gene of the incriminated HAdV-7 strain, we analysed the whole genome sequence variation and viral growth of the Wuhan isolates. Knowledge of a link between VA RNA II genetic variation and HAdV-7 virulence may help us understand the function of this gene and take effective measures to control HAdV-7 prevalence.th of January and 21st of February, 2015. Health officials, medical staff, epidemiologists, and laboratory technicians were designated as an epidemic prevention team to investigate and control the outbreak. The names, onset times, clinical symptoms and informed consent for sample collection and subsequent research usage of the students were documented by trained medical staff and epidemiologists. Copies of the case records of the hospitalized students were provided by the corresponding doctors. All the protocols used in these investigations were approved by the Institutional Review Boards of the Centre of Disease Control and Prevention of China, and the Ethics Committee of Wuhan General Hospital.An outbreak of ARD was reported in a college in Wuhan city, Hubei Province, between the 20Bordetella pertussis, Legionella pneumophila, Chlamydophila pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, and Mycoplasma pneumoniae were detected with Seeplex®RV12 ACE and Seeplex® Pneumabacter detection kits . After confirmation of HAdV infection, real-time PCR primers for HAdV serotypes 7, 11, 14, and 55 were used for serotyping . Pure Link Viral RNA/DNA Mini Kits  were used to extract nucleic acids. Suspected pathogens, such as adenovirus, coronavirus, parainfluenza virus, influenza A virus, influenza B virus, respiratory syncytial virus A, rotyping .The samples of nasopharyngeal swabs were inoculated into A549 cells cultured in DMEM. The viruses were isolated by repeated freeze-thaw and preserved in -80°C. Primer sequences used for the amplification of hexon, fibre and VA RNA genes have been published elsewhere . The who5’-TGCAAAGAGCTCTAACGGGG-3’ and R: 5’-CTTCCAAGTACTCGCCCTCC-3’. Reference genes VA RNA II and E1 were detected using primers VA-F: 5’-GAGCCAGTGCTGCGTCAA-3’, VA-R: 5’- TTAGAAACGTCGCGGAAA-3’; and E1-F: 5’-CCACCTACGCTGCACGAT-3’, E1-R: 5’- ACCTCTGCCGCTTTCCAC-3’, respectively. KAPA SYBR FAST qPCR Kits  were used for amplification.Viral RNA was extracted using an Ultrapure RNA extraction kit . The primers used for the detection of L1 52/55K mRNA were F: 2 genome copies) were propagated in human A549 cells cultured in 12-well plates and were collected 6, 9, 12, 18, 21, 24, 36, 48, and 72 h after infection. The cultures were subjected to repeated freezing and thawing thrice and centrifuged at 12000×g for 10 min. Then, 200 μl of supernatants were used for DNA extraction using Pure Link Viral DNA Mini Kits and virus titres were determined by detecting the number of viral E1 gene copies by real-time PCR. The E1 gene fragment was cloned into the T vector  and diluted to 0.5 × 102–0.5 × 109 copies/μL as standard samples.Viruses  and were analysed using Student’s th of January and 21st of February, 2015  developed the illness and 169 of them were admitted to Wuhan General Hospital between the 20ry, 2015 . The aveAccording to case reports, patients presented typical respiratory infection symptoms, such as fever (92.3%), cough (83.4%), sore throat (62.1%), swelling of tonsils (47.9%), and a runny nose (26.0%). Among them, 92 patients (54.4%) were diagnosed with upper respiratory infection, 77 (45.6%) with pneumonia , and 121Of the 4113 students, 739 were identified as HAdV-7-positive by real-time PCR. The ratio among hospitalized patients was 76.9% (130 out of 169). Twenty viral strains were isolated from hospitalized positive patients and cultured in the laboratory; after which hexon, fibre, and VA RNA genes were amplified and sequenced. Alignments of these genes of the isolated 20 virus produced 100% similarity.Next, we used the BLASTn tool to find the strains with closest relation to the sequenced hexon, fibre, and VA RNA genes. Both hexon and fibre genes showed 100% identity with the HAdV-7 isolate CQ1198 (GenBank accession number JX625134), which belongs to the HAdV-7d type , revealeThen, we chose the Z9/WH strain (GenBank No. KX897164) of these Wuhan isolates as representative to make whole-genome sequencing, and the comparison result with strain CDC228 revealed that the Z9/WH strain had some other but insignificant mutations except for VA RNA gene .Phylogenetic trees based on hexon, fibre, and VA RNA gene sequences were constructed respectively. As all sequences were identical, one sample Wuhan strain (KU351170) was taken as representative. And the Z9/WH strain (GenBank No. KX897164) was chosen for the whole-genome sequence and DNA polymerase gene analysis  and other HAdV-7 strains found in China in recent years. The DNA polymerase gene and whole genome sequence of HAdV-7 found in China in recent years were located within the same branch but with a little difference. And the VA RNA I sequences from all serotype 7 strains clustered together and the sequences were highly conserved except for occasional mutations in the termination region , pneumonia (45.6%) and 8 ICU (intensive care unit) cases. To understand the reason behind this large scale outbreak, genomic variations were detected in our article.Hexon and fibre capsid proteins are the main antigenic determinants of HAdV serotypes. In a previous work on two other HAdV-7 outbreaks, we found that most genes had few or no mutations besides those in VA RNA genes, which displayed a 12-bp deletion . TherefoHow the lost T in the VA RNA II terminal region played influence on HAdV-7 is still unknown. Interestingly, the HAdV VA RNA genes are located right in front of the L1 52/55K gene , which pIn summary, we report here an uncommon outbreak of HAdV-7 among college students in Wuhan, China, in 2015. Sequence analysis revealed several missing T in the VA RNA II terminal region. This mutation, in turn, caused up-regulation of an adjacent gene, L1 52/55K. The latter promoted faster growth of the HAdV-7 strain, thus providing a causal link between the VA RNA II gene and a severe clinical outcome. Taking into account the phylogenetic results on the conserved nature of HAdV-7 hexon and fibre genes in China in recent years, VA RNA genes represent a new determinant factor for HAdV-7 virulence upon infection. However, further work, such as functional verification of the missing T bases by constructing a deletion mutant of strain CDC228 or reverse mutant of the WH strain, should be done to elucidate the function of the VA RNA II gene and its relationship to the increased growth capacity of the HAdV-7 strain.S1 AppendixFig A. Phylogenetic analysis of HAdV-7 hexon and fibre genes. Fig B. Phylogenetic analysis of DNA polymerase gene and VA RNA I gene of HAdV-7. Fig C. Sequence alignment of HAdV-7 VA genes. Fig D. Sequence alignment of VA II gene of HAdVs.(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Sporisorium scitamineum, which can cause severe economic losses in sugarcane industry. The infection depends on the mating of bipolar sporida to form a dikaryon and develops into hyphae to penetrate the meristematic tissue of sugarcane. In this study, we set to isolate bacterial strains capable of blocking the fungal mating and evaluate their potential in control of sugarcane smut disease. A bacterial isolate ST4 from rhizosphere displayed potent inhibitory activity against the mating of S. scitamineum bipolar sporida and was selected for further study. Phylogenetic analyses and biochemical characterization showed that the isolate was most similar to Pseudomonas guariconensis. Methanol extracts from minimum and potato dextrose agar (PDA) agar medium, on which strain ST4 has grown, showed strong inhibitory activity on the sexual mating of S. scitamineum sporida, without killing the haploid cells MAT-1 or MAT-2. Further analysis showed that only glucose, but not sucrose, maltose, and fructose, could support strain ST4 to produce antagonistic chemicals. Consistent with the above findings, greenhouse trials showed that addition of 2% glucose to the bacterial inoculum significantly increased the strain ST4 biocontrol efficiency against sugarcane smut disease by 77% than the inoculum without glucose. The results from this study depict a new strategy to screen for biocontrol agents for control and prevention of the sugarcane smut disease.Sugarcane smut is a fungal disease caused by  Sporisorium scitamineum is a devastating disease in sugarcane growing areas globally, which results in considerable loss of sugar yield  metabolite(s) with potent activity in inhibiting the sexual mating of S. scitamineum without killing fungal haploid cells. Greenhouse trials showed that Pseudomonas sp. ST4 could be a promising biocontrol agent for effective control of sugarcane smut disease.The pathogen produces two types of haploid sporida, which mate together to form a dikaryon, and then develops into hyphae to penetrate the bud scales of the sugarcane plant and infect host meristematic tissues . Therefo2HPO4, 4.5 g KH2PO4, 2 g (NH4)2SO4, 2 g mannitol, 2 g glycerol, 0.2 g MgSO4 7H2O, 5 mg FeSO4, 10 mg CaCl2, and 2 mg MnCl2, pH 7.0) or on solid medium containing 1.8% (wt/vol) agar at 28°C  at 28°C or on solid medium containing 1.8% (wt/vol) agar  or minimal medium broth  agar . Potato S. scitamineum, the PDA plate was cut into separated slices (0.6 cm in width). An aliquot of 1 μl bacterial culture was added on one end of the agar slice, and then the mixture of S. scitamineum haploid cells MAT-1 and MAT-2 strain was spotted (0.5 μl of OD600 ≈ 1.5) on the slice at progressively further distances from the loaded sample. LB medium was added in the same way as bacterial culture as a negative control. The plates were incubated at 28°C for 2 days, until the white hypha in the negative control grew to reach the edges of the slice. The candidate isolates inhibiting hyphal growth but allowing haploid growth were selected for further analysis.Over 3000 bacterial isolates were obtained from soil samples collected from five provinces of China, including Guangdong, Henan, Xinjiang, Jiangxi, and Guangxi, following a method described previously with minor modifications . Briefly600 ≈ 1.5) were evenly spotted at about 1 cm distance on PDA plates. The plates were incubated at 28°C for 48 h, and bacterial cells were removed by peeling off the agar surface containing the bacterial colonies. The remaining PDA medium was melted at around 70°C, and mixed with fresh PDA medium at 1:1 ratio before pouring into a new petri dish. After solidification, a mixture of S. scitamineum haploid cells MAT-1 and MAT-2 was spotted (0.5 μl of OD600 ≈ 1.5) on the plate. A mixture spotted on a fresh PDA plate was used as a negative control. The plates were incubated at 28°C for 1–2 days, until white hypha appeared in the control plate.As the inhibitory activity was hardly detectable in liquid culture supernatants, we produced ST4 extracellular metabolites using PDA agar plates. Briefly, strain ST4 cells  as indicated. For testing the effect of different sugars and derivatives on the inhibitory activity of strain ST4 against the sexual mating of Sporisorium scitamineum haploid strains MAT-1 and MAT-2 were, respectively, inoculated into 100 ml YEPS liquid medium at 28°C, shaking at 200 rpm for 2 days. Strain ST4 was cultured in 100 ml LB medium at 28°C, shaking at 200 rpm for 12 h. Cells were collected by centrifuge at 3000 rpm for 10 min. Collected cells were washed twice with PBS buffer and re-suspended in PBS buffer (OD600 ≈ 3.0). The above three fungal and bacterial strains were mixed at 1:1:1 ratio with or without glucose , and an aliquot of 1 mL of treatment was inoculated by injection to the seedling (around four leaves) stem base of sugarcane cultivar ROC22, which is susceptible to S. scitamineum. Sugarcane seedlings were inoculated with the mixture of two fungal haploid strains as a positive control, and same amount of PBS buffer as a negative control. Thirty seedlings were used in each treatment and the trial was performed twice.TM DNA Purification Kit following the manufacturer’s protocol . Primers used for amplification of the 16S rRNA gene were 27F and 1492R with conditions determined by gyrB), RNA polymerase β subunit (rpoB), and RNA polymerase subunit D (rpoD) genes were performed as described , pH 7.0] for 2 min, air-dried and observed by using transmission electron microscope (Hitachi H7650).Phenotypic characterization was carried out according to the minimal standards proposed by S. scitamineum. A total of 68 isolates showed different levels of inhibitory effect on the hyphal growth of S. scitamineum, but for most of them, this effect was due to killing or stopping the growth of haploid cells rather than direct interference of the fungal sexual mating, which was confirmed by using ST4 metabolite plate assay (data not shown). Bacterial isolate ST4, which showed a strong activity to inhibit the hyphal growth of mixed S. scitamineum cells  grew poorer than the cells at further distance. These fungal cells at a, b, and c were re-inoculated onto a fresh PDA plate, and the results showed that the haploid cells grew up normally  and the mixture cells produced hyphal cells , indicating that the diffusible metabolites from strain ST4 didn’t kill the fungal cells.More than 3000 bacterial isolates were isolated from the rhizospheres of sugarcane, vegetables, bamboo, maize, and many other plants from five provinces in China, including Guangdong, Henan, Xinjiang, Jiangxi, and Guangxi. These bacterial isolates were spotted onto PDA plates for screening of bacterial antagonists against the sexual mating of Figure 1C), and the colony morphology was similar to the corresponding haploid cell lines grew on the ST4 metabolite plates . In contrast, the MAT-1 and MAT-2 mixture produced white hyphal cells on the control E. coli metabolite agarose plate . In another set of experiments, we showed that haploid cell lines MAT-1 or MAT-2 on the ST4 metabolite plate  grew up in a way similar to the same cell lines on fresh PDA plates . To distinguish whether ST4 metabolites exactly affect mating of bipolar sporida or hyphal growth of the resultant dikaryon, we used visualized MAT-1 and MAT-2 haploid cells, respectively, . To determine whether ST4 affects hyphal growth, we transferred hyphae from PDA after mating to new plates of PDA and PDA+ST4 metabolites, respectively. Results showed that hyphae became fragmented, fractured, and vanished after ST4 treatment . Taken together, the above results indicate that strain ST4 produces a metabolite(s) capable of inhibiting not only the sexual mating of S. scitamineum, but also the growth of haploid cells or dikaryon hyphae.The ability of strain ST4 on inhibition of fungal mating was further tested by using metabolite plate of ST4, as we were not able to detect visible inhibitory activity using its culture supernatants. ST4 inhibitory metabolite plates were prepared by growing strain ST4 on PDA plate, removing bacterial cells after 2 days, melting the remaining medium at around 70°C and mixing with fresh PDA medium at 1:1 ratio. The control agarose plates were prepared in the same way by using strain DH5α to replace strain ST4, which did not show any inhibitory activity against the fungal mating , and could also substantially increase the production of mating inhibitory compounds .In our preliminary study, we found that PDA medium was superior to LB, MM, and PA media for detection of inhibitory activity of strain ST4 against the fungal sexual mating. Given that PDA differs from PA by containing glucose, we tested whether addition of glucose to various media could improve production of mating inhibitory molecule(s) by strain ST4. The results showed that supplementation of glucose at a final concentration of 20 g per liter in LB, MM and PA media could improve hyphal growth of the fungal pathogen .Similarly, we then tested whether other sugar or sugar derivatives could promote strain ST4 to produce inhibitory compounds, using glucose as a positive control. The results showed that only glucose could support strain ST4 to produce inhibitory compounds against the fungal sexual mating, whereas sucrose, maltose, fructose, and starch had no any effect . In a separate experiment, we found that addition of glucose alone in the same concentration range as in Figure 5 without strain ST4 could not inhibit the fungal mating (data not shown), suggesting that glucose might be used as a substrate by strain ST4 in production of inhibitory compounds.Dosage analysis showed that the inhibitory activity of strain ST4 was enhanced along with the increased concentration of glucose from 1% till 8%, but no inhibitory activity could be detected when glucose concentration was at 0.5% . In contrast, addition of strain ST4 and glucose in the fungal inoculation mixture blocked smut disease symptom development . Consistent with the results of plate bioassay , addition of strain ST4 alone without glucose in the inoculation mixture had no effect on biocontrol of the smut disease . As expected, the sugarcane seedling showing smut disease symptoms grew poor than the control plants without inoculation and those treated with strain ST4 and glucose .The biocontrol potential of strain ST4 was determined under greenhouse conditions. The results showed that the smut disease symptoms became visible at about 3 months after inoculation of mixed fungal cell lines MAT-1 and MAT-2 (Table 2) showed that smut incidence was maximum in pots in which sugarcane sets were planted after inoculation of the fungal lines MAT-1 and MAT-2, following by the pots in which sugarcane sets was inoculated with the mixture containing MAT-1, MAT-2, and strain ST4, but the smut incidence was significantly reduced by about 65% when the MAT-1/MAT-2/ST4 mixture was inoculated in the presence of glucose .Quantitative analysis of the disease rate , P. taiwanensis (99.22%), P. monteilii (99.14%), P. plecoglossicida (99.07%), P. mosselii (99%), P. entomophila (99%), and P. putida (98.22%) . We further determined the partial gene sequences of three other housekeeping genes, i.e., gyrB, rpoB, and rpoD, of strain ST4, which share 94.25, 94.71, and 93.69% identity to their counterparts in P. guariconensis, respectively .The strain ST4 was isolated from vegetable rhizosphere in Shantou city, Guangdong province, China. Its colonies on LB plate are beige, translucent, convex, round, and smooth with entire margins. Electron micrograph showed that cells are straight rods, motile by means of polar flagella, with wrinkle cell surfaces . Positive for arginine dihydrolase, urease, oxidase, and catalase; but negative for nitrate reduction, aesculin hydrolysis, indole and β-galactosidase production. In Biolog GN III plates, strain ST was positive in assimilation of D-glucose, D-fructose, glycerol, L-arginine, L-histidine, L-pyroglutamic acid, L-serine, L-aspartic acid, L-glutamic acid, L-lactic acid, citric acid, D-malic acid, α-keto-glutaric acid, L-malic acid, bromo-succinic acid, β-hydroxy-D, D-gluconic acid, L-butyric acid, propionic acid and amino-butryric acid. However, strain ST4 could not utilize sucrose, D-turanose, stachyose, gelatin, N-acetyld-D-glucosamine, N-acetyld-β-D-glucosamine, α-hydroxy-butyric acid, N-acetyl neuraminic acid, D-cellobiose, D-galactose, gentiobiose, lactose, maltose, D-mannitol, D-mannose, D-melibiose, L-rhamnose, D-trehalose, D-saccharic acid, D-glucose-6-phosphate, β-methyl-D-glucosamine, mucic acid, D-salicin, 3-methyl glucose, D-sorbitol, glycyl-L-prolin, α-keto-butyric acid, N-acetyl-D-galactosamine, D-raffinose, D-arabitol, m-inositol, D-aspartic acid, acetoacetic acid and formic acid. Reactions to dextrin, D-serine, tween 40, D-fucose, L-fucose, inosine, D-fructose-6-phosphate, L-alanine, pectin, D-galacturonic acid lactone, D-glucuronic acid, glucuronamide, methyl pyruvate, D-lactic acid methyl ester and acetic acid were variable (Table 3). The above morphological and biochemical properties of strain ST4 are reminiscent but not identical with those of P. guariconensis strain PCAVU11 (Table 3). In combination of the phylogenetic analysis and the results of biochemical test, we recommend assigning strain ST4 as P. guariconensis.Further biochemical tests showed that strain ST4 is Gram negative and strictly aerobic. Optimum temperature for growth is 28°C, grew at 44°C, pH range for growth is 5–10, could grow with 0–5% NaCl in LB broth. The DNA G+C content was 63.56 mol%. A diffusible fluorescent pigment is produced on King’s B medium, which is the same as Rhizosphere is a region full of benificial or deleterious microorganisms, and is the first defense line from the attack by soilborne pathogens . TherefoS. scitamineum, while allowing growth of its haploid cells. We further showed that strain ST4 provided effective biocontrol against sugarcane smut disease under greenhouse conditions. To circumvent the environmental hazards and residual problems associated with application of chemical fungicides, plant and fungal extracts, which acted through inhibition of fungal mycelial growth and teliospore germination, were tested to control sugarcane smut disease with some effect , and glucose was essential for strain ST4 to produce the fungal mating-inhibitory compounds and to control the sugarcane smut disease . This is intriguing as among a few saccharides tested, including sucrose, maltose, and fructose, none of them could support strain ST4 to produce the fungal mating-inhibitory compounds. Given that the inhibitory activity of strain ST4 was increasing along with the increment of glucose concentration, we reasoned that glucose or its metabolites could be used for biosynthesis of the anti-mating compounds by strain ST4. Purification and characterization of the inhibitory compounds and elucidation of the corresponding biosynthetic pathway shall shade a light to dissolve this intriguing puzzle.As widely known, the recognized mechanisms of biocontrol include competition for niches or substrates, production of inhibitory compounds or cell wall degrading enzymes, and induction of systemic resistance . Our resP. guariconensis based on genotypic, phenotypic and chemotaxonomic analyses. P. guariconensis is a newly identified bacterial species and only one report, which was on taxonomic characterization , possibly reducing the compatibility of the haploid cells, implicating the possible reason of the actions of ST4 on the sexual mating of S. scitamineum. Furthermore, different components extracted from strain ST4 showed complicated actions on S. scitamineum, including suppression of the hyphal growth such as component S-2-6, and inhibition of the sexual mating, such as component S-2-7 (Supplementary Figure In general, this is the first report on biocontrol of sugarcane smut disease through antagonism against the sexual mating and hyphal growth of SL and LZ conceived and designed the experiments; JZ and YT helped to identify strain ST4; SL, NL, YmC, ZL, LL, ML, and YfC helped to perform the molecular work; FH and SC helped to analyze the active components produced by ST4; JZ and LZ revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Yet, the contribution of marsh oiling to landscape-scale degradation and subsequent land loss has been difficult to quantify. Here, we applied advanced remote sensing techniques to map changes in marsh land cover and open water before and after oiling. We segmented the marsh shorelines into non-oiled and oiled reaches and calculated the land loss rates for each 10% increase in oil cover (e.g. 0% to >70%), to determine if land loss rates for each reach oiling category were significantly different before and after oiling. Finally, we calculated background land-loss rates to separate natural and oil-related erosion and land loss. Oiling caused significant increases in land losses, particularly along reaches of heavy oiling (>20% oil cover). For reaches with ≥20% oiling, land loss rates increased abruptly during the 2010–2013 period, and the loss rates during this period are significantly different from both the pre-oiling (p < 0.0001) and 2013–2016 post-oiling periods (p < 0.0001). The pre-oiling and 2013–2016 post-oiling periods exhibit no significant differences in land loss rates across oiled and non-oiled reaches (p = 0.557). We conclude that oiling increased land loss by more than 50%, but that land loss rates returned to background levels within 3–6 years after oiling, suggesting that oiling results in a large but temporary increase in land loss rates along the shoreline.The 2010 BP Deepwater Horizon (DWH) oil spill damaged thousands of km Coastal wetlands provide a myriad of important ecosystem services, including flood mitigation, pollution removal, carbon sequestration, wildlife habitat and recreational opportunities, but they are threatened by an array of human activities, both directly by dredging, channelization and construction, and indirectly by sea level rise and reduced sediment input. Intertidal ecosystems, particularly salt marshes, are resilient to physical disturbances, which has been attributed to their high productivity ,2,3 and 2 yr-1) [2 of intertidal wetland area from 1932–2010 (∽62 km2 yr-1) [For nearly two centuries, human activities in the northern Gulf of Mexico have altered natural hydrologic regimes and changed the magnitude of system perturbations beyond salt marsh resilience thresholds ,10, resu2 yr-1) . Since t2 yr-1) ,14,15,16m2 yr-1) . A combim2 yr-1) .2 of wetland area per year since 1932 [Barataria Bay, a rapidly eroding abandoned delta where aggradation is no longer keeping pace with the effects of eustasy and subsidence , perhapsnce 1932 ,20. Yet,nce 1932 ,21. Episnce 1932 ,24,25,263 of crude oil into the Gulf before being capped on July 15 [The largest oil spill in U.S. history occurred in the Gulf of Mexico on April 20, 2010, when an explosion on the Deepwater Horizon (DWH) offshore drilling unit released 780,000 m July 15 . Oil was July 15 . Oiling  July 15 ,29,30, c July 15 ,31. Expo July 15 ,33, or p July 15 . Soil st July 15 ,34,35. R July 15 ,26,36,37Barataria Bay was among the areas most heavily impacted by oil following the DWH spill . The thrSpartina alterniflora dominated marshes were extensively degraded and that vegetation classes converted to an open water class along oiled shorelines at more than double the rate of non-oiled shorelines from 2010–2012. In comparing pre-oiling (2009–2010) shoreline recession rates, Rangoonwala et al. [Three recent studies used remote sensing techniques to examine the impacts of oiling on salt marshes of Barataria Bay on a landscape-scale ,42,43. B2 m-1 yr-1) were calculated to standardize the loss rates for varying shoreline lengths, and to provide results that can be easily compared with future assessments of marsh loss along the shoreline in the Louisiana Coastal Zone. The rationale for examining the land loss rates at three year time intervals derived from the temporal response patterns documented in prior research [In this study, our objectives were to: a) map changes in land loss along the shoreline in a bay affected by the DWH oil spill for three time periods: before, 3 years after, and 6 years after the spill, b) determine if rates of land loss were significantly different before and 3 and 6 years after the spill, and c) quantify the impact of oiling on reach-scale and bay-wide loss rates, controlling for temporal variability in natural background erosion rates. Land loss rates per unit shoreline  and oiled reaches, and land loss rates were calculated to determine if loss rates were significantly different for oiled and non-oiled reaches, and for pre- (3 years before oiling) and post-oiling  time periods. Last, we calculated background land loss rates for oiled reaches, using a combination of oiled (pre-spill) and non-oiled (pre- and post-spill) shoreline loss rates, to estimate the magnitude of oil-related land losses that are not attributable to temporal variability in background loss rates.2 in northern Barataria Bay, Louisiana , and consists of 41 km2 of marsh area and 133 km of marsh shoreline  (The study area covers approximately 197 kml banks) . BaratarSoils in the lower Barataria Basin form on sediment and are tidally redistributed in the lower basin . Soils  imagery and published in Peterson et al. . AVIRIS https://www.digitalglobe.com) and Aerometric Inc. (http://gis.aerometric.net/dirlists.htm) for the four dates used in this study (High resolution (0.30–0.64 m) orthorecitifed image datasets were acquired from DigitalGlobe .Image acquisition time could affect the amount of water mapped due to tides, therefore, image data captured at or below mean low water (MLW) are ideal and acquisition at 1–2 feet (0.31–0.62 m) above MLW is acceptable for the northern Gulf of Mexico ,54. Here2) in the southern Barataria Basin resulted in frequently overlapping onshore transects and created shoreline reaches that were variable in length  in m yr-1:slr is calculated as the land area loss (a) over the segmented longshore reach length (l) divided by the number of years between image acquisitions (t). The time intervals (t) between imaging dates were 3.4 (2006–2010), 3.6 (2010–2013) and 2.5 (2013–2016) years. We performed reach-level pair-wise T-tests, and then summarized the p-values for each oiling category to determine if post-oiling land loss rates were significantly different from the pre-oiling rates (To account for the variable lengths of the created longshore reaches (15–172 m), we normalized the total land loss by the reach length to get a standardized loss rate (ng rates .blr) for a given oiling category (j) for the post-oiling periods as:slr for post-oil  and pre-oil  periods for non-oiled reaches (oil cover = 0):Post-spill land loss rates were higher for all shoreline reaches, including non-oiled reaches, presumably due to normal erosion forces affecting all reaches, such as wave energy, currents, tides and sediment supply. Additionally, storm surge from Hurricane Isaac, which made landfall in the study area in August 2012, likely contributed to increased land losses during the first post-oiling period (2010–2013). Therefore, we estimated background loss rates (1 = 1.36) was calculated by dividing the post-oiling (2010–2013) period slr (0.36 m2 m-1 yr-1) by the pre-oiling slr (0.26 m2 m-1 yr-1) for non-oiled reaches. The oil-related loss rate (olr) is the difference between the observed slr and the blr. All three loss rates were converted into an area loss rate by multiplying the loss rate by the length of shoreline for each oiling category.For instance, the post-oiling (2010–2013) change coefficient  exhibited no detectable oiling (i.e. oil cover = 0) from July 2010—April 2011 , S2 Fig.2 yr-1 (0.49 m2 m-1 yr-1) to 153,676 m2 yr-1 (1.44 m2 m-1 yr-1) from 2010–2013, and decreased to 85,388 m2 yr-1 (0.58 m2 m-1 yr-1) from 2013–2016 , however, there were no statistical differences in slr between 2010–2013 and the pre-oiling period  or the 2013–2016 period  (2/yr) of the total land loss during 2010–2013, despite accounting for 62% of the shoreline length (2 yr-1) and 61%  of the total land loss during the 2006–2010 and 2013–2016 periods, respectively  more than doubled the losses from the pre-oiling period (2006–2010) , and the013–2016 . For non= 0.249) . Even th= 0.249) . Land loe length . In compectively , which iectively . Furtherectively .2 yr-1 of land loss, or 62% of the total land loss for the post-oiling period 1 (2010–2013). In comparison, the land losses from the pre-oiling period  and post-oiling period 2  are more similar to the relative length of the shorelines. Reaches with ≥50% oiling contributed 37% of the land loss during the post-oiling period 1 (2010–2013), while accounting for 21% of the shoreline length  . Further . Furthee length .2 m-1 yr-1 for oiled reaches, and range from 0.06–1.11 and 0.05–0.98 m2/m/yr over the two consecutive post-oiling periods , more than 80% of which are attributable to the 30–70% oiled reaches , if interior marshes were included in the analysis. A more appropriate comparison of non-oiled near-shore marsh loss rates is with the bay islands of Barataria Bay, due to their similar biogeomorphic profiles, which are comprised of low, relatively flat monotypic  marsh platforms behind 30–50 cm natural levees at the marsh edge [-1 and 2013–2016: 1.23% yr-1) are comparable to the marsh island area loss rates of 1.5–1.6% reported in Turner et al. [-1) are also similar to the rates reported previously in specific study sites of Barataria Bay (0.80–1.38 m yr-1) [Our reported rates  are somewhat higher than what Barras et al.  (0.90%) rsh edge ,43. Our  m yr-1) ,26,38.Several recent studies have used remote sensing techniques to assess the impacts of oiling on salt marsh vegetation ,64,65,66Spartina patens than those dominated by S. alterniflora. In both field and mesocosm experiments, S. alterniflora has exhibited a greater recovery rate than Juncus roemerianus, indicating a higher tolerance limit for oil contamination [S. alterniflora than J. roemerianus after 18 months under heavy oiling conditions [S. alterniflora dominated marshes were extensively degraded following the DWH spill, losing 15%  cover along oiled shorelines, suggesting that marsh degradation may have been worse if the oil-impacted marshes were dominated by other species i.e. (J. roemerianus or S. patens).Heavy oiling has complex and interactive effects on the structural and physiological traits of marsh macrophytes that likely influence recovery success ,40,68. Pmination ,69. Livenditions . Recentlnditions  reported2 yr-1), we determined 52,521 m2 yr-1 of land was lost due to oiling, increasing the land losses by 52% over the background rate.For heavily oiled shorelines (>50% oil fraction), we report loss rates 2.7 times greater  than that of non-oiled shorelines for the first 3-years after oiling. This magnitude of impact from oiling is consistent with observations of land loss on Barataria Bay marsh islands from Turner et al.  and fromTwo marsh erosion processes, driven by heavy oiling, were presumably contributing to the accelerated rates of land loss that we observed. Exposure to heavy oiling obstructs critical, adaptive mechanisms for reducing oxygen stress in anoxic soils , and forThe influence of other factors, including: oiling characteristics ,79,80,81p ≥ 0.095)  . Land loWe provided a landscape-scale, bay-wide quantification of land loss, while documenting the return to background erosion rates 3–6 years after oiling. Yet, several obvious questions remain unaddressed, such as what is the relative importance of lateral erosional forces from wave action in comparison to vertical forces (i.e. reduced sediment accretion and subsidence) in Barataria Bay? Wilson and Allison,  estimateThe impact the use of marsh treatments had on bay-wide land losses is still largely unknown. Clean-up and treatment efforts affect the recovery process, both positively and negatively ,83. AggrFinally, we showed that land losses increased significantly for the first three years after oiling, followed by a return to background erosion levels after three years. To date, this process of returning to background rates of erosion remains unexplained, and should be addressed in future research. Hester et al.  reported2 yr-1, in the first post-oil period (2010–2013), more than 80% of which are attributable to the 30–70% oiled reaches. No statistical difference was observed for any reach oiling categories from pre-oiling and post-oiling period 2 (2013–2016). Oiling increased land loss by more than 50%, but land loss rates returned to background levels within 3–6 years after oiling, suggesting that oiling results in a large but temporary increase in shoreline loss.We examined the relative land loss rates of oiled shoreline reaches compared to non-oiled reaches of Barataria Bay over three consecutive time periods. Oiling increased total land losses by 52,521 mblr) derived from pre-oiling land loss patterns and post-oiling land loss in non-oiled reaches. Our calculation of blr assumes that the ratio between the loss rate in the pre-oil and post-oil period is the same under non-oiled conditions for all shoreline orientations and locations in the bay. Wave modeling could be included in future analysis to control for changes in shoreline orientations, wind direction and fetch between periods.We attempted to control for effect of other erosional forces  on land loss by calculating the background rates Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S1 FigExamples of shoreline zones (green) and the onshore transects (black lines) that were used to segment the shoreline into reaches. For shorelines that were relatively linear (A), the transects segmented the shoreline into 100m reaches. However, for undulating shorelines (B), onshore transect direction was irregular and at times overlapping, creating shorter onshore reaches. Additionally, transect generation on small islands (C) commonly resulted in inconsistent reach lengths, and in some cases, required manual removal of selective transects.(TIF)Click here for additional data file.S2 Fig(ZIP)Click here for additional data file.