Lean-type Pekin duck is a breed gained through long-term selection and great effort has been exerted to understand the mechanisms underlying increased muscle yields. However, the genes involved in Pekin duck embryonic breast muscle development have not been explored to date. In this study, we investigated gene expression profiles in Pekin Duck embryonic breast muscle at hatched day 13 (E13), E19, and E27 using RNA-seq. In total, we produced 519,312,178 raw reads resulting in 497,348,158 high-quality reads after filtering. The mapping, distribution of reads along annotated genes, and consistency across replicates demonstrates the high quality of the RNA-seq data used in this study, allowing us to continue with the downstream analysis. Significantly fewer differentially expressed genes (DEGs) were identified between E13 and E19 (203 DEGs) compared to E27 and E19 . Many DEGs highly expressed in E19 are involved in metabolic processes and cell division. KEGG analysis showed many pathways associated with fat development were significantly enriched for DEGs highly expressed in E27. These results provide a basis for the further investigation of the mechanisms involved in Pekin duck embryonic breast muscle development. MYF5), muscle-specific regulatory factor 4 (MRF4), myoblast determination protein (MYOD), and myogenin [SIX1-SIX6), eyes absent (EYA), and muscle enhance factor 2C (MEF2C) act as skeletal myogenesis regulators [Skeletal myogenesis is a multistep process in which mesodermal precursor cells are selected to form myoblasts that are withdrawn from the normal cell cycle and subsequently differentiate into myotubes , 2. Thesgulators . ObviousMUSTN1, MSTN, IGF1, and FOXO3 genes with breast muscle development in postnatal Pekin duck [Pekin duck is world-famous for its fast growth, but its skeletal muscle yield is lower than that of other high lean yield ducks . To incrkin duck –13. In akin duck . Based okin duck . HoweverHere, we constructed three mRNA libraries from Pekin duck embryonic breast muscle at E13, E19, and E27. Gene expression profiles were determined via RNA-seq data and subsequent bioinformatics analysis in this study. We further investigated the roles of differentially expressed genes (DEGs) for the development and regulation of Pekin duck embryonic breast muscle. The results presented here, in combined with our previous results, provide further insights into the molecular mechanism underlying breast muscle development in Pekin duck.® Spectrophotometer. The total RNA was used for construction of cDNA libraries and subsequent sequencing according to Xu et al. [Three full-sib Pekin duck embryos per time point at stage E13, E19 and E27 were collected in sterile physiological saline immediately after removal from the hatching eggs. Breast muscle samples were frozen in liquid nitrogen and stored at −80°C. Total RNA was isolated from all samples using the RNAiso plus kit  following the manufacturer's instructions. The RNA quality was analyzed by 1.0% agarose gel electrophoresis and spectrophotometric absorption at 260 nm in a Nanodrop ND-1000To ensure the RNA-seq data was of high enough quality for downstream analysis, we carried out adapter trimming and filtering of the raw reads to decrease the data noise. Specifically, we filtered the raw reads to remove: i) adapter contamination, ii) reads in which unknown bases were more than 5% of the read, and iii) low quality reads .http://www.ensembl.org/Anas_platyrhynchos/Info/Index) using SOAPaligner/SOAP2 software [We mapped the high-quality reads to the duck reference genome  method . The detWe first defined the significantly expressed genes as the genes expressed in all of the three time points  with their p-values and correct p-values (FDR) less than 0.05. Then, we performed clustering analysis of the genes to identify their expression tendencies by using hierarchical clustering schemes . The detGenes differentially expressed between two time points were identified using the method described by Audic and Claverie . Genes wqRT-PCR was performed using the SYBR PrimeScript RT-PCR Kit  with SYBR Green dye to validate the RNA-seq results as previously described . PrimersData described in this study is available in the NIH Short Read Archive (SRA) under accession number SUB1749540.Financial support of this work was provided by “the earmarked fund for China Agriculture Research System ”All sample collection and subsequent experiments were approved by the Ethical and Animal Welfare Committee of Beijing, China. Birds were slaughtered using the electric shock method followed by the oral bloodletting method within 30 seconds to ameliorate their suffering.RNA-seq data was produced using RNA obtained from Pekin duck embryonic breast muscle samples at E13, E19 and E27. In total, 519,312,178 raw reads were obtained , including 171,658,529 for E13, 179,524,180 for E19, and 168,132,911 for E27. After filtering, a total of 497,348,158 high-quality reads  remained and were used for downstream analysis, including 164,148,468 for E13, 171,960,228 for E19, and 161,239,462 for E27. Overall, ~95.77% of raw reads were defined as high-quality reads and the average GC content of high-quality reads was 50.44% . Much hiThe high-quality reads were then mapped to the duck reference genome. The basic statistical results are summarized in To demonstrate the positional relationship between the mapped reads and annotated genes, we classified the mapped reads into several types such as the reads located in coding sequence (CDS), introns, intergenic regions, and noncoding exons . The majLarger RNA molecules must be fragmented into smaller pieces (200–500 bp) to be compatible with RNA-seq deep-sequencing technology during the RNA-seq library construction. Compared to other methods, RNA fragmentation provides more even coverage along the gene body, while reducing coverage at the 5′ and 3′ ends . TherefoThe consistency of the gene expression results across replicates at each time point is an important index for the reliability of RNA-seq data . In thisAPQ4, ACTA1, GAS2, TFCP2L1, and ENPP6) across replicates using qRT-PCR and RPKM approaches. The results showed no significant differences in gene expression based on the RPKM values  to 6.07 (GAP43). When comparing the E27 and E19 samples, 2,797 DEGs were detected, 1,437 of which displayed increased expression in the E19 samples, compared to 1,360 displaying increased expression in the E27 samples (LOC101803953) to 9.13 (KLHL38). As the fastest growth rate for Pekin duck embryonic breast muscle occurs at E19 [The mainobjective of this study was to identify DEGs involved in Pekin duck embryonic breast muscle development. The general landscape of DEGs is demonstrated in  samples . Log2 Fos at E19 , DEGs wide facto standard in gene functionality description, is used widely for functional annotation and enrichment analysis [Gene Ontology (GO), the analysis . Here, wanalysis . IV) DEGKEGG (Kyoto Encyclopedia of Genes and Genomes) is a knowledge base for systematic analysis of gene functions, linking genomic information with higher order functional information . We perfOf the pathways enriched for DEGs highly expressed at E27 compared to E19, the pathway containing the most DEGs was the metabolic pathway (167 genes), indicating a high level of metabolic activity in Pekin duck breast muscle at E27. In addition, many pathways were associated with fat development, including PPAR signaling pathway, adipocytokine signaling pathway, glycerophospholipid metabolism, glycerolipid metabolism, and fatty acid degradation. Previous reports have identified decreased myofibres and satellite cell mitotic activity in the breast muscle of late-term turkey embryos , indicatA number of pathways enriched for DEGs highly expressed at in E19 compared to E27 were involved in cell development, such as DNA replication, cell cycle, biosynthesis of amino acids, gap junction, non-homologous end-joining, and base excision repair, consistent with previous reports indicating Pekin duck breast muscle undergoes its quickest growth phase at E19 .The KEGG enrichment analysis allows us to identify DEGs highly expressed at E27 that play a role in fat development, providing candidate genes for further investigation of the mechanisms underlying skeletal muscle and fat development at E27.In the current study, we identified many DEGs involved in duck muscle development and fat deposition, including NR1H3, ADIPOR2, SOCS3, ACADM, FABP5, RXRA, FABP3, STK11, CD36, CYP8B1, ACSL4, ACSL1, UBB, HADHB, FABP4, PRKAA2, HADHA, ACSBG2, ACOX1, ACADL, ECI2, mTOR, CYP27A1, NFKB1, PRKAB2, and SLC27A1.in vivo through the Akt/mTOR pathway [To validate the expression of the above-mentioned DEGs, we compared the expression levels of the five most important DEGs involved in muscle development and fat deposition  using qRT-PCR . mTOR  and KEGG pathways  were significantly enriched for DEGs highly expressed at E19 compared to the other two time points. In addition, many pathways  enriched for DEGs highly expressed at E27 compared to E19 are involved in fat development. Finally, many DEGs involved in skeletal muscle development and fat deposition, including mTOR, NFKB1, NR1H3, FABP4, and SLC27A1, were identified in this study.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.


BTG3 transfectants exhibited lower viability and higher apoptosis than the control in both time- and dose-dependent manners. BTG3 overexpression up- regulated the protein expression of Cyclin E, p16, p27, NF-κB, p38α/β, XIAP, Bcl-2, ATG14 and p53, but down-regulated the mRNA expression of MRP1, BCRP, and mTOR in SW480 and SW620 cells. BTG3 overexpression inhibited tumor growth of SW620 cells by suppressing proliferation and inducing apoptosis. It was suggested that down-regulated BTG3 expression might be considered as a marker for colorectal carcinogenesis. BTG3 overexpression might reverse the aggressive phenotypes and be employed as a potential target for gene therapy of colorectal cancer.Here, we found that down-regulated expression of BTG3 might be positively correlated with colorectal carcinogenesis and its overexpression suppressed proliferation, glycolysis, mitochondrial respiration, cell cycle progression, migration, and invasion, and induced apoptosis, senescence and differentiation in SW480 and SW620 cells. After treated with cisplatin, MG132, paclitaxel and SAHA,  Although the incidence and mortality have been decreasing in the past two decades due to the alteration in risk factors and colorectal cancer screening, it challenges the health of the human being , 2. TherBTG3 is distributed to human chromosome 21q21.1 and transcriptionally produces two variants with 132-nucleotide deletion by alternative splicing. Its encoding protein is found to inhibit cell proliferation, cell cycle progression and induce differentiation as a tumor suppressor [via the interaction with checkpoint kinase 1 (CHK1). BTG3-mediated maintenance of genomic stability requires its Lys63 ubiquitination and CHK1 activation [BTG3 abrogation might enhance bone morphogenetic protein- induced ectopic bone formation [BTG3 expression was attributable to its promoter methylation in breast, lung, prostate or renal, hepatocellular, and gastric cancer tissues or cells [BTG3 knockdown promoted proliferation and tumorigenicity of ovarian clear cell carcinoma. Our group also demonstrated that BTG3 overexpression inhibited proliferation, migration, invasion and tumor growth, induced S/G2 arrest, differentiation, autophagy, apoptosis and chemosensitivity of gastric cancer cells [ppressor . BTG3 cappressor . BTG3 alppressor , and witppressor . BTG3 cativation , 8. BTG3ormation . The dowor cells –15. The or cells . Yanagidor cells  showed ter cells .BTG3 mRNA and protein in CRCs, and compared them with clinicopathological parameters of cancers. Its promoter methylation was measured in colorectal cancer cells and tissues. Finally, the in vivo effect of BTG3 overexpression on tumor growth was determined in nude mice bearing CRC cells.To clarify the roles of BTG3 in colorectal carcinogenesis and subsequent progression, we investigated the effects of BTG3 overexpression on cell proliferation, glucose metabolism, apoptosis, senescence, differentiation, invasion and migration of CRC cells and screened the expression of the phenotype-related genes. Moreover, we examined the expression of BTG3 mRNA in DLD-1, Colo 201, Colo 205, and KM-12 . BTG3 overexpression decreased the expression of MRP1, BCRP, and mTOR in SW480 and SW620 cells .As shown in Figure y Figure , p<0.05.s Figure , p<0.05.s Figure , p<0.05.BTG3 transfectant were subcutaneously transplanted into immune-deficient nude mice. As shown in Figure BTG3 transfectants by ruling, weighting and capacity measurement respectively (p<0.05). BTG3 protein was strongly expressed in the cytoplasm of the transfectant, compared with the control. The transfectants showed a low expression of ki-67 (a marker for proliferation) and a strong signal of TUNEL (a marker for apoptosis) in comparison to the control . Then, we used Gaedcke's, Hong's, Skrzypczak's and TCGA's dataset to perform bioinformatical analysis and found that BTG3 mRNA expression was higher in colorectal normal mucosa than adenoma or cancer  than that in cancer Figure , p<0.05.M Figure , p<0.05.M Figure , p<0.05.M Figure , p<0.05.r Figure , p<0.05.BTG3 protein was distributed in the cytoplasm of colorectal epithelium, infiltrating inflammatory cells, macrophages, hepatocytes, lymphoid follicle, adenoma, and cancers Figure . BTG3 exBTG3 expression was positively correlated with depth of invasion and differentiation of CRCs Table , p<0.05.BTG3 expression was decreased in CRC, compared with adjacent NNM by Western blot and real-time RT-PCR, even followed by LCM. These findings suggested that down-regulated BTG3 expression contributed to the malignant transformation of colorectal epithelial cells, consistent with these documents [BTG3b mRNA was found in colorectal cancer and NNM although BTG3a and 3b were detectable in lung cancer cells and tissues [BTG3 mRNA expression, which was reported in gastric, breast, renal and prostate cancer cells [BTG3 methylation and mRNA expression in CRC, it was speculated that BTG3 methylation was partially responsible for its silenced expression. In contrast, more frequent methylation of BTG3 was found in colorectal mucosa than cancer, similar to its mRNA, while versa for BTG3 immunopositivity. The paradoxical phenomenon might be hypothesized to correlate with BTG3 methylation, weaker BTG3 expression in cytosol-marginal stromal cells, and BTG3 degradation by ubiquitin-proteasome system [In line with the results of oncomine analysis, we found that ocuments , 18, 19. tissues . After ter cells –12, 18. e system  as demonin vitro [in vivo [BTG3 mRNA had about 1.83 fold higher in primary than metastatic cancer of colon by cDNA microarray. Exogenous BTG3 protein suppresses the levels of MMP-2 and PAI-1 expression in lung cancer cells [via Wnt/β-catenin signaling pathway [Here, BTG3 overexpression enhanced ALP level of CRC cells, supporting the positive link between BTG3 overexpression and differentiation of CRCs. There appeared BTG3 overexpression in deeply-invasive cancer, while BTG3 overexpression suppressed proliferation, migration, invasion, tumor growth, glycolysis and mitochondrial respiration with the induction of apoptosis or senescence as the reports about gastric cancer , and esoin vitro , and inh[in vivo , 18. In [in vivo  found ther cells . Reporteer cells , 18, 19.er cells . Another pathway . TherefoBTG3 transfectant showed a higher chemosensitivity to cisplatin, MG132, paclitaxel and SAHA, which was positively correlated with a high apoptotic induction. Additionally, BTG3 overexpression results in the mRNA hypoexpression of chemoresistance- related genes, such as MRP1, BCRP, and mTOR. MDR1 and BCRP belong to efflux transporters of the ATP-binding cassette family and pumps many foreign substances out of cells [3K /Akt/mTOR pathway in some malignancies [Furthermore, of cells . Chemoregnancies . Taken t3K, Beclin 1, LC-3B, and β-catenin in BTG3 might underlie the molecular mechanisms of the differences in the biological behaviors of SW480 and SW620, even their BTG3 transfectants. In apoptosis, Bcl-2 can interact with Bax on the mitochondrial membrane to suppress the apoptosis [BTG3 transfectants was demonstrated to not correlate with BTG3-induced apoptosis in CRC cells. According to LC-3B expression, the effects of BTG3 on autophagy were different between SW420 and SW680, and positively linked to Beclin 1 expression, indicating that BTG3-induced autophagy was dependent on Beclin-1 and belonged to canonical pathway. Here, BTG3 overexpression was found to activate NF- κB pathway by up-regulating its transcriptional activity and expression, but its biological effects should be deeply investigated.Although both SW480 and SW620 cells are separated from primary and lymph node metastasis foci of the same CRC patients, the inconsistent expression of Cyclin B1, AIF, PIpoptosis . XIAP mipoptosis . The up-in vivo and vitro appeared a low proliferation in BTG3 transfecants, BTG3 overexpression resulted in G1 arrest in SW480 cells and S arrest in SW620 cells. Reportedly, Cyclin E and D1 activate Cdks and induce the transition between G1 and S phase [1 arrest [1/S transition. BTG3-mediated cell cycle arrest might be due to the overexpression of above-mentioned 3 proteins.Although there  S phase . Cyclin  S phase . Therefo1 arrest , 30. ActBTG3 expression was found to positively associate with favorable prognosis of gastric and ovarian cancers , 16. Lv BTG3 is partially responsible for its down- regulated expression. BTG3 overexpression might in vitro and vivo reverse the aggressive phenotypes of CRC cells and be employed as a target molecule for gene therapy.In summary, down-regulated BTG3 expression might be positively correlated with the malignant transformation of colorectal epithelial cells and should be considered as a good biomarker for colorectal carcinogenesis. Promoter methylation of 2 at 37°C. SW480 and SW620 cells transfected with pcDNA3.1-BTG3 or pcDNA3.1 vector, and selected by G418 according to the manufacturer's instructions . We treated CRC cells with 5-aza-20-deoxycytidine , cisplatin (a platinum- containing DNA crosslinker), MG132 (a proteasome inhibitor), paclitaxel (a mitotic inhibitor), and SAHA (a histone deacetylase inhibitor). All cells were pelleted by centrifugation, rinsed with PBS, and subjected to protein and RNA extraction.CRC cell lines were kindly presented by Prof. Miyagi, Clinical Research Institute, Kanagawa Cancer Center, Japan. They were cultured in RPMI 1640  and DMEM (WiDr) medium added with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin in an atmosphere of 5% COCell counting Kit-8  was used to measure cell viability. Briefly, 4000cells/well were seeded on 96-well plate, which was added with 10 μL CCK-8 solution at different time points. After the incubation for 3 h, optical density was read without cover at 450 nm.The cells were harvested, washed by PBS and fixed in cold ethanol for 4 h at −20°C. After washed by PBS, the cells were immersed with 1mL RNase (0.25 mg/mL) at 37°C for 1 h. The cells were pelleted, resuspended in propidium iodide  and incubated in the dark for 30 min. Finally, flow cytometry was carried out to detect PI signal.KeyGEN Biotech) to examine apoptosis as the instructions recommend.Flow cytometry was carried out with PI and FITC-labeled Annexin V  and Biorad protein assay kit respectively. ALP activity was calculated as U per μg of protein.β-galactosidase staining kit  was employed to indicate the senescence. The protocol was executed as recommended by the kit instruction.Both oxygen consumption rates and extracellular acidification rates were measured in XF media  under basal conditions and in response to mitochondrial inhibitors, 1 mM oligomycin and/or 100 nM rotenone + 1 mM antimycin A (Sigma) on XF-96 Extracellular Flux Analyzers (Seahorse Bioscience).The luciferase reporter assay was performed using Dual-Luciferase®Reporter Assay System  as recommended by the manufacturer. NF-κB-mediated gene transcription activity was determined by the ratio of pGL3- NF-κB luciferase activity, which was normalized to Renilla luciferase activity of the control plasmid.Colorectal cancers (n=484), adenoma (n=126), adjacent NNM (n=475), metastatic foci in lymph node metastasis (n=150) and in liver (n=21) were collected from the surgical resection in the Affiliated Hospital of Kanagawa Cancer Center (Japan) between 1999 and 2008. The patients with CRC were 281 men and 203 women . Forty-five cases of CRCs and paired NNM were collected from the Shengjing Hospital of China Medical University (China) and frozen in −80°C until use. None of the patients received chemotherapy, radiotherapy or adjuvant before surgery. All of them provided consent for use of tumor tissues for clinical research. Ethical Committee of our hospital and Kanagawa Cancer Center approved the research protocol. We followed up the patients by consulting their case documents or calling.All tissues were fixed in 10% neutral formalin and prepared for paraffin-embedded block and 4 μm-thick section. These sections were subjected to hematoxylin-and-eosin staining for routine pathological examination, including tumor size, depth of invasion, lymphatic and venous invasion, lymph node metastasis and liver metastasis. The staging for each colorectal cancer was evaluated according to tumor-node-metastasis system . Histolo6 cells per mouse to axilla (n=10 mice /group). For each tumor, measurements were made using calipers, and tumor volumes were calculated as follows: width2× length ×0.52. After anesthetization, the mice were photographed, and sacrificed. Tumors was weighted and cupped in PBS for the volume. After that, the part of tumors were fixed in 4% paraformaldehyde, and embedded in paraffin for following experiments.Locally bred female 6-week-old Balb/c nude mice were used for implantation. They were maintained under specific pathogen-free condition. Housing and all procedures were approved by the committee for animal experiments guidelines on animal welfare of our hospital. Xenografts were established by subcutaneous injection of 1× 10Colorectal cancer and glandular epithelial cells were dissected from 8-μm-thick and stained tissue slides  by an Arcturus Veritas™ laser capture microdissection system (Life Technologies) for total RNA extract using Pure Link RNA Mini Kit (Ambion).Total RNA was extracted from colorectal cancer cells or tissues using QIAGEN RNeasy mini kit (Germany), and subjected to cDNA synthesis using AMV transcriptase and random primers . According to the Genbank, oligonucleotide primers were designed as described previously . GeneralBTG3 as described previously [We extracted genomic DNA from colorectal cancer cells and tissues using QIAamp DNA Mini kit (QIAGEN). DNA was modified chemically with sodium metabisulphite using EZ DNA Methylation-Gold Kit (ZYMO Research). The bisulfite-modified DNA was amplified by using primer pairs that specifically amplify either methylated or unmethylated eviously .Denatured protein was separated on an SDS-polyacrylamide gel and transferred to Hybond membrane. The membrane was in turn blocked in 5% skim milk, primary Table  and secoTwo-mm-in-diameter TMA was prepared using a Tissue Microarrayer . TMA was consecutively cut into 4-μm-thick sections, which were deparaffinised, rehydrated, and subjected to antigen retrieval in target retrieval solution .The sections were quenched with 3% hydrogen peroxide and incubated with 5% bovine serum albumin. The sections were incubated with the rabbit antibody against BTG3  or ki-67 , and then treated with the anti-rabbit conjugated to horseradish peroxidase  antibody. Binding sites were visualized with 3, 3'-diaminobenzidine. After counterstained with Mayer's hematoxylin, the sections were dehydrated, cleared and mounted. Omission of the primary antibody was used as a negative control.BTG3 protein was positively localized in the cytoplasm, while ki-67 in nucleus. One hundred cells were randomly selected and counted from 5 representative fields of each section blindly by two independent observers (Zheng HC and Zhao S). The inconsistent data were determined for final agreement by both persons. The expression positivity was graded and counted as follows: 0 =negative; 1 = 1-50%; 2 = 50-74%; 3 ≥75%. The staining intensity score was graded as follows: 1 = weak; 2 = intermediate; and 3 = strong. The scores for BTG3 positivity and staining intensity were multiplied to obtain a final score, which determines their expression as .In Situ Apoptosis Detection Kit (Millipore) was used according to the recommendation. Omission of the working strength TdT enzyme was considered as a negative control.Cell apoptosis was assessed using TUENL, a method that is based on the specific binding O-TdT to the 3-OH ends of DNA. Therefore, ApopTag Plus Peroxidase BTG3 was analyzed using Oncomine (www.oncomine.org), a cancer microarray database and web-based data mining platform for a new discovery from genome-wide expression analyses. We compared the differences in BTG3 mRNA level between colorectal mucosa and adenoma/cancer. All data were log-transformed, median centered per array, and standard deviation normalized to one per array.The individual gene expression level of Spearman’s correlation test and student t test were used to analyze the rank data and compare the means respectively. Kaplan-Meier survival plots were generated and comparisons between survival curves were made with the log-rank statistics. Cox's proportional hazards model was employed for multivariate analysis. SPSS 10.0 software was applied to analyze all data and p<0.05 was considered statistically significant.


Immunohistochemistry is a widely available technique that is less challenging and can provide clinically meaningful results quickly and cost-efficiently in comparison with other techniques. In addition, immunohistochemistry allows for the evaluation of cellular localization of proteins in the context of tumor structure. In an era of precision medicine, pathologists are required to classify lung cancer into specific subtypes and assess biomarkers relevant to molecular-targeted therapies. This review summarizes the hot topics of immunohistochemistry in lung cancer, including (i) adenocarcinoma vs squamous cell carcinoma; (ii) neuroendocrine markers; (iii) ALK, ROS1, and EGFR; (iv) PD-L1 (CD274); (v) lung carcinoma vs malignant mesothelioma; and (vi) NUT carcinoma. Major pitfalls in evaluating immunohistochemical results are also described. In an era of precision medicine, immunohistochemistry plays a critical role in the classification of tumors into subtypes and for assessing biomarkers for timely and accurate therapeutic decision-making ,2,3,4,5.Lung cancer is the leading cause of cancer-related deaths worldwide, regardless of gender. It is categorized into two main groups: small cell lung carcinoma  and non-SCLC . Accumulating evidence suggests that lung cancer represents a group of histologically and molecularly heterogeneous diseases ,15,16,17The 2015 World Health Organization (WHO) classification was recently modified based on newly identified molecular profiles and druggable genetic alterations in lung cancer . In partWhen possible, differential diagnosis between adenocarcinoma and SqCC is beneficial because targetable driver genetic alterations are mostly identified in adenocarcinoma, and inappropriate drugs need to be avoided for patients with SqCC. Before the 2015 WHO classification, the definitions of adenocarcinoma and SqCC were based on their morphological features with or without mucin staining. Adenocarcinoma was defined as carcinoma with an acinar/tubular structure or mucin production, whereas SqCC was defined as carcinoma with keratinization or intercellular bridges. In the current classification, a solid carcinoma without glandular structures or mucin production, but with immunohistochemical positivity for “adenocarcinoma markers”, i.e., TTF-1 (NKX2-1) and/or Napsin A, is diagnosed as an adenocarcinoma. Similarly, a solid carcinoma without keratinization or intercellular bridges, but with immunohistochemical positivity for “SqCC markers”, such as p40, CK5/6, and TP63 (p63), is diagnosed as SqCC. These modifications using immunohistochemical evaluations have markedly minimized the proportion of NSCLC diagnosed as large cell carcinoma .There exist several pitfalls in differential diagnosis between adenocarcinoma and SqCC ,21,22. WIn the 2015 WHO classification , the catNeuroendocrine differentiation of lung tumors is orchestrated by complex pathways as concisely displayed in A recent study demonstrated that INSM1 was positive in 94.9% of SCLCs and 91.3% of LCNECs, compared with 74.4% and 78.3% with the combined panel of the three neuroendocrine markers . Thus, INSM1 was suggested to be more useful than the individual or combined use of CHGA, SYP, and NCAM1 for the diagnosis of lung HGNETs . INSM1 aVarious somatic genetic alterations in tyrosine kinase have emerged as druggable molecular targets by tyrosine kinase inhibitors (TKIs), particularly in lung adenocarcinoma . With a ALK-rearranged lung adenocarcinoma, comprising 4‒5% of lung adenocarcinomas, is clinicopathologically characterized by a TTF-1 cell lineage, an acinar structure with mucin/signet-ring cell morphology, non-/light-smoking history, and young onset [ALK-rearranged NSCLC [ALK rearrangement, in addition to conventional fluorescence in situ hybridization (FISH) [ALK-rearranged NSCLC. Therefore, patients with ALK-positive NSCLC, as confirmed by immunostaining using clone D5F3, are candidates for ALK inhibitor treatment. Of note, immunohistochemistry using the ALK D5F3 antibody appears to be not only a candidate but also the most important test for ALK testing. There are several studies suggesting the superiority of the ALK immunohistochemistry with the D5F3 antibody compared with ALK FISH at predicting response to ALK inhibitors [ng onset ,33,34,35ed NSCLC ,37,38. In (FISH) . Accordin (FISH) . Recentlhibitors ,42,43,44ALK-rearranged lung cancers show cytoplasmic ALK staining. Intracellular mucin vacuoles are frequently observed in ALK-rearranged adenocarcinoma; thus, inadequate cytoplasmic immunostaining can cause these to be carelessly missed  of lung adenocarcinomas [ROS1-rearranged adenocarcinoma is clinicopathologically characterized by solid growth with signet-ring cells or a cribriform morphology with abundant extracellular mucus, and typically occurs in younger non-smoking females [ROS1 rearrangement confirmed by FISH assays [ALK-rearranged NSCLCs, immunohistochemistry provides high sensitivity and specificity for the detection of ROS1 rearrangements. Immunohistochemical assay using the specific rabbit monoclonal antibody clone D4D6 is a cost-efficient and widely available method for screening patients with ROS1-rearranged NSCLCs [ROS1 rearrangement need to be used as an external positive control [ROS1 fusion. Adenocarcinomas with CD74-ROS1 fusion, which is the most frequent fusion gene, usually shows globular cytoplasmic ROS1 immunoreactivity, whereas adenocarcinomas with EZR-ROS1 fusion usually show membranous immunostaining [ALK-rearranged adenocarcinomas, an intracellular mucin vacuole is frequently observed in ROS1-rearranged adenocarcinomas; therefore, inadequate immunostaining should not be missed. It should be also noted that benign hyperplastic pneumocytes and macrophages frequently show weak ROS1 immunostaining [ROS1 rearrangement, as confirmed by both immunohistochemical and FISH analyses [rcinomas ,46,47. R females . ClinicaH assays . As withd NSCLCs ,51. Howe control . Furtherstaining . Similarstaining . Furtherstaining . Currentsubset 1‒% of lungEGFR mutation is one of the most common driver mutations in lung adenocarcinoma, and EGFR-mutated adenocarcinoma is characterized by East Asian ethnicity, female gender, and non-/light-smoking history [EGFR-mutated lung adenocarcinoma typically shows nuclear TTF-1 immunoreactivity and hobnail cell morphology. In addition, adenocarcinoma with micropapillary morphology has a higher frequency of EGFR mutations than adenocarcinoma without this morphology [EGFR mutations are divided into four major types: point mutations in exon 18, deletions in exon 19, insertions in exon 20, and point mutations in exon 21. Approximately 90% of EGFR mutations in NSCLCs involve in-frame deletions in exon 19 and the point mutation L858R in exon 21. These mutations, particularly exon 19 deletions, are associated with a superior and prolonged clinical response to EGFR TKIs [EGFR mutation-specific antibodies, recognizing a 15-bp deletion in exon 19 (clone: 6B6) and an L858R point mutation in exon 21 (clone: 43B2), have been developed [EGFR mutations due to its low sensitivity.The  history . Patholorphology . In the GFR TKIs ,58. EGFReveloped . HoweverPD-L1 CD274) is an immune modulator that promotes immunosuppression by binding to PD-1 (PDCD1). PD-L1 on the surface of tumor cells inhibits an immune-mediated attack by binding to PD-1 on cytotoxic T-cells ,61. Alth is an imSpecific immunohistochemical assays for different PD-1/PD-L1 inhibitors have been designed to estimate sensitivities to these treatments . CurrentAs an external positive control for PD-L1, human tonsils, placenta, or PD-L1-positive cell lines can be used. In addition, at least 100 viable tumor cells need to present in one PD-L1-immunostained slide to determine the percentage of PD-L1-positive cells. Of note, the careful observation of both hematoxylin-eosin (HE)-stained slides and immunostained slides is required to correctly evaluate PD-L1 positivity, as PD-L1 can be immunostained in inflammatory cells, including macrophages and lymphocytes. When PD-L1-positive macrophages and/or lymphocytes exist around PD-L1-negative tumor cells, misinterpretation should be carefully avoided. In addition, tumor cells with cytoplasmic granular immunostaining, but without membranous staining, should not be misinterpreted as positive ,3.Malignant mesothelioma (MM) is a rare and fatal malignant tumor arising from mesothelial cells. Malignant mesothelioma is one of the important tumors that need to be distinguished from lung cancer while diagnosing lung cancer. Asbestos exposure is the main risk factor for developing MMs, which are generally classified into three major histologic subtypes: epithelioid 60–80%), sarcomatoid (<10%), and biphasic (10–15%). Epithelioid MM is the most common subtype and shows a relatively better prognosis than sarcomatoid or biphasic MM [0–80%, saCDKN2A (p16) deletions, identified by FISH, have recently emerged as potential indicators of MM. Loss of nuclear BAP1 immunostaining is often observed in MMs, particularly the epithelioid/biphasic subtype, but not in active mesothelial proliferations. However, BAP1 loss is relatively uncommon in the sarcomatoid subtype. Therefore, BAP1 immunohistochemistry has a relatively high specificity, but low sensitivity [CDKN2A deletion is observed more frequently in the sarcomatoid subtype than in the epithelioid/biphasic subtype [Reactive mesothelial proliferations occasionally mimic MMs, because reactive mesothelial proliferations sometimes exhibit cellular and structural atypia typically observed in MMs. Therefore, differential diagnosis between MM and reactive mesothelial proliferation is often difficult. The loss of BAP1, confirmed by immunohistochemistry, and homozygous sitivity ,88,89. I subtype .Lung carcinomas can also show pseudomesotheliomatous spreads. As no immunohistochemical marker is completely specific for each type of tumor, the International Mesothelioma Interest Group recommends at least two mesothelial and two carcinoma markers, in addition to cytokeratins, to be included in the differential diagnosis between MM and carcinoma. Mesothelial markers include calretinin (nuclear and cytoplasmic staining), podoplanin , CK5 or CK5/6 (cytoplasmic staining), and WT1 (nuclear staining) . For difCollectively, immunohistochemistry plays an essential role in the differential diagnosis of MM; thus, the appropriate selection of immunohistochemical markers is essential. Morphological, clinical, and radiological features are also needed for correct diagnosis.NUT (NUTM1) gene on chromosome 15q14 and one of the other partner genes. The NUT gene is fused to the bromodomain family member BRD3 on chromosome 19p13.1 (comprising 70% of NUT carcinomas), BRD4 on chromosome 9q (6%), or the other partner genes. The methyltransferase NSD3 on chromosome 8q11.23 was newly recognized as a fusion partner of NUT [NUT. This contrasts with lung SqCCs, which are characterized by complex and multiple cytogenetic alterations [NUT carcinoma has been recently recognized as a subtype of lung cancer in the 2015 WHO classification . Becauseosome 9q %, or ther of NUT ,97,98,99r of NUT . Althougosome 9q %, or theerations . In termClinically, NUT carcinoma shows an extremely aggressive behavior with dismal prognosis, and a median overall survival of 2.2 months. NUT carcinoma occurs with no predominance of gender, and affects people of any age and smoking history, although it was originally reported in young individuals ,94,101. Morphologically, NUT carcinoma is characterized by sheets and nests of small- to intermediate-sized monotonous, primitive-appearing tumor cells A, with tImmunohistochemical detection of the nuclear NUT protein facilitates a rapid and cost-effective diagnosis of NUT carcinoma, with the aid of highly specific and sensitive monoclonal NUT antibodies . NUT carImmunohistochemical techniques play critical roles as diagnostic and screening tools for lung cancer. However, the limitations of immunohistochemistry should be fully apprehended to avoid inappropriate results from immunohistochemical assays ,107. The


Carotenoid cleavage dioxygenase (CCD) genes are involved in the biosynthesis of SLs and are identified and characterized in many plants. However, the function of CCD genes in tobacco remains poorly understood. In this study, two closely related genes NtCCD8A and NtCCD8B were cloned from tobacco (Nicotiana tabacum L.). The two NtCCD8 genes are orthologues of the tomato (Solanum lycopersicum) carotenoid cleavage dioxygenase 8 (SlCCD8) gene. NtCCD8A and NtCCD8B were primarily expressed in tobacco roots, but low expression levels of these genes were detected in all plant tissues, and their transcript levels significantly increased in response to phosphate limitation. NtCCD8A and NtCCD8B mutations were introduced into tobacco using the CRISPR/Cas9 system and transgenic tobacco lines for both ntccd8 mutant alleles were identified. The ntccd8a and ntccd8b mutant alleles were inactivated by a deletion of three nucleotides and insertion of one nucleotide, respectively, both of which led to the production of premature stop codons. The ntccd8 mutants had increased shoot branching, reduced plant height, increased number of leaves and nodes, and reduced total plant biomass compared to wild-type plants; however, the root-to-shoot ratio was unchanged. In addition, mutant lines had shorter primary roots and more of lateral roots than wild type. These results suggest that NtCCD8 genes are important for changes in tobacco plant architecture.Strigolactones (SLs) are a class of phytohormones that regulate plant architecture.  Striga lutea [Strigolactones (SLs) are new types of plant hormones that regulate many aspects of plant growth and developmental processes. Strigol was the first characterized SL, which was exuded from cotton roots and induces the germination stimulant of ga lutea . SLs arega lutea ,3,4. SLsga lutea ,6,7. Conga lutea ,9,10.rms) in pea (Pisum sativum), more axillary growth (max) in Arabidopsis, decreased apical dominance (dad) in petunia (Petunia hybrida), and dwarf (d) or high-tillering dwarf (htd) in rice (Oryza sativa) [As a newly discovered endogenous hormones, SLs play a role in controlling above- and below-ground plant architecture ,13,14,15 sativa) ,20,21,22 sativa) ,24,25. S sativa) ,29,30,31 sativa) ,35,36,37CCD7 and CCD8 genes have been identified in the SLs synthetic pathways of several plant species, including Arabidopsis, pea, rice, tomato, saffron, Phelipanche aegyptiaca, maize, apple, poplar and petunia [SlCCD8 and SlCCD7 down-regulated by RNA interference reduced strigolactone concentrations in tomato [CCD8 were also characterized in potato, maize, chrysanthemum and kiwifruit [Arabidopsis, pea, petunia and rice [Strigolactones are a group of carotenoid-derived molecules ,38. More petunia ,52,53,54n tomato ,46. Orthiwifruit ,56,57,58iwifruit . MAX1 haand rice ,60,61,62and rice .Nicotiana tabacum L.) is widely cultivated worldwide as a non-food crops, and is also an important model plant species for basic biological research. To date, the genes involved in the SLs signaling system have been identified in Arabidopsis, pea and rice [NtCCD8 genes from tobacco were cloned and their role in SLs biosynthesis and tobacco plant growth and development was characterized via the targeted knockout using the CRISPR/Cas9 system.Tobacco  tobacco genome database (http://solgenomics.net/organism/Nicotiana_tabacum/genome) using a tBLASTn search. Two pu-tative coding sequences (mRNA_112966_cds and mRNA_104099_cds) from tobacco CCD8 genes were identified from the tBLASTn search against the SGN database. These nucleotide sequences were used to design the specific primers for the full-length CCD8 coding sequences, which were amplified from tobacco root tissues. The tobacco CCD8 genes have two open reading frames (ORF) that are 1668 bp and 1671 bp in length and encode 556 and 557 amino acid proteins, respectively. Based on their high identity to SlCCD8, tobacco CCD8 proteins were named NtCCD8A and NtCCD8B. The nucleotide sequences of both NtCCD8 genes from the tobacco genome database were aligned with the ORF sequences, and the two NtCCD8 genes were predicted to have six exons  and nutrient deficiency. NtCCD8A and NtCCD8B expression in root tissues increased under phosphate starvation, and NtCCD8A expression was six-fold higher than that of NtCCD8B  in NtCCD8A and NtCCD8B was selected and this sequence is located adjacent to protospacer adjacent motif (PAM), which is essential for Cas9 to recognize and cleave the target site  in the ORF and created loss-of-function mutants  method. 5-Deoxystrigol, one of SLs, was detected in the root extracts of wild-type plants, but it not in the root extracts of ntccd8 mutants  levels in the leaves of ntccd8 mutants were significantly lower than in wild-type plants, which had twice as much in total chlorophyll content that the mutants  is an allotetraploid (2n = 48 resulting from both parents chromosomes sets being present in the gametes) that resulted from a Nicotiana sylvestris (2n = 24) and Nicotiana tomentosiformis (2n = 24) hybridization [NtCCD8A and NtCCD8B, encode the tobacco carotenoid cleavage dioxygenase NtCCD8A and NtCCD8B, respectively. When their amino acid sequences were aligned with those of N. sylvestris (NsyCCD8) and N. tomentosiformis (NtomCCD8), respectively, results showed that NtCCD8A originated from NtomCCD8 and NtCCD8B originated from NsyCCD8  content were observed in ntccd8 mutants compared to wild-type plants  and of the homozygous transgenic NtCCD8 knockout lines were surface-sterilized and grown on Murashige and Skoog (MS) Basal Medium  containing 30 g/L sucrose and 8 g/L agar. One-month-old seedlings were transferred to pots with a peat moss to perlite mixture ratio of 3:1 and kept in a greenhouse under long-day conditions  or subject to natural season conditions.Seeds of tobacco  and 1-Naphthaleneacetic acid (NAA) . For phoNicotiana tabacum BX genome database (http://solgenomics.net/organism/Nicotiana_tabacum/genome) using the tomato SlCCD8 and potato PhCCD8 protein sequences as query subjects in a basic local alignment search tool  with default settings. Two full-length NtCCD8 coding sequences were amplified from tobacco root tissue cDNA by PCR, using specific primers . Successfully amplified PCR products were cloned into the pEASY-Blunt Zero Cloning Vector  and sequenced.Two NtCCD8-like translated nucleotide sequences were obtained from the Sol Genomics Network (SGN)  primers  and the Arabidopsis, tomato, petunia and tobacco were performed in Clustal X version 1.8 using default parameters . To construct the phylogenetic tree, CCD8, CCD7, CCD4 and CCD1 protein sequences from several plants were aligned. An unrooted phylogenetic tree based on the CCD proteins alignment was constructed in MEGA Version 4.0 , using the Neighbor-Joining (NJ) method with parameters of pairwise gap deletion and 1000 bootstraps.Multiple protein sequence alignments of CCD8 orthologs from NtCCD8A and NtCCD8B in tobacco, root, stem, leaf and flower tissues of 70-day-old tobacco plants were collected, immediately frozen in liquid nitrogen, and kept at −80 °C until RNA extraction.To detect the expression patterns of ® Plant Mini Kit and treated with the RNase-free DNase kit  according to the manufacture’s protocols. Total RNA concentration was quantified on a NanoDrop ND-1000 Spectrophotometer . Total RNA (1 μg) was used for first-strand cDNA synthesis using M-MLV Reverse Transcriptase . Quantitative reverse transcriptase PCR (qRT-PCR) was performed using SYBR Premix-Ex Taq  and gene specific primers . To detect mutations, the genomic regions including the Cas9/gRNA target sites were amplified by PCR using the specific primers . For the analysis of seedling root growth, 7-day-old seedlings of wild-type and mutant plants were grown in small square Petri dishes (12 cm × 12 cm) on solid MS medium. After ten days, the phenotype of seedlings was photographed, and the number of lateral roots was counted manually and the main root lengths were measured using ImageJ 1.49 software (http://rsb.info.nih.gov/ij/index.html).Plant height, leaf number, internode length and stem perimeter were measured in 20-day-old, 40-day-old, 50-day-old, 60-day-old and 90-day-old wild-type and target knockout plants. Plant height was defined as the plant length from the soil level to the the highest leaf. Nodes and internodes were numbered from the first leaf node at the bottom of the plant to its upper parts, and internode length was calculated as the ratio of plant height to internode numbers. Stem perimeter was measured at the third internode counting from the apex. Shoot branching was monitored and analyzed based on the number and length of branches longer than 0.5 cm. The number and length of shoot branching were also counted and measured. The dry weight of above- and below-ground biomass was also measured. All the above-mentioned traits were described at least six plants from each independent line. The roots of tobacco mutants were collected from three individual plants and imaged with a scanner. The total root length, surface area and volume were then analyzed using the WinRhizog. The supernatant was gently transferred to another set of 5 mL microcentrifuge tubes and the pellets was re-extracted with another 2 mL of ethyl acetate. The supernatants were combined and the ethyl acetate was evaporated to dryness under vacuum conditions. The dried fractions were re-suspended in 150 μL of acetonitrile: water  solution, filtered, and subjected to ultra-performance liquid chromatography tandem spectrometry (UPLC-MS/MS) analysis.For SLs analysis, roots from mutant and wild-type plants were harvested 50 day after transplanting, frozen in liquid nitrogen and freeze-dried. The procedure of SL extraction was performed as previously described . Freshly−1 flow rate and at 50 °C. Mobile phase’s initial composition (5% B) was maintained for 2 min, increased from 5 to 50% B in 8 min, from 50 to 90% in 1 min, held at 90% for 0.1 min, and returned to the initial conditions using a 0.2-min gradient. Finally, the column was equilibrated for 2.8 min before the next injection start. Injection volume was 15 μL. The mass spectrometer was operated in positive electrospray ionization mode (ESI+). The spectra were recorded using target-sim mode (331.153 m/z) and full scan mode, covering a mass range from 100 to 1000 m/z. The resolution was set at 70,000. SLs analysis were carried out using the method as previously reported with minor modifications . A quadrParameters of for the ion source were set as follows: the mass spectrum (MS) spray voltages 3.5 kV, capillary temperature 320 °C, probe temperature 100 °C, sheath gas pressure 35 arb, auxiliary air pressure 5 arb. Date acquisition and analysis were performed using the Thermo Xcalibur software (Thermo Fisher Scientific).To extract chlorophyll, leaves located at identical positions along the main stem were sampled from three wild type and three mutant plants, and analyzed as described previously . Leaves NtCCD8 genes editing were analyzed and evaluated as previously reported [The potential off-target effects resulting from reported .t-test. GraphPad Prism 5.0  was used for plotting data and imaging.Statistical analysis was performed on all data using Student’s


Lippia citriodora (L. citriodora) on rats subjectedto chronic constriction injury (CCI) of sciatic nerve and possible mechanisms of actions. We examined the protective effects of ethanolic extract of  In this experimental study, the extract was administered 50, 100 and 200 mg/kg, Intraperitoneally(I.P) from the surgery time for 14 consecutive days. The changes in the spinal cord levels of apoptotic factors, microgliaand astroglia markers during the time course of study were assessed by western blotting on days 3, 7 and 14 post-CCI. CCI rats developed neuropathy evident from a marked mechanical allodynia, cold allodynia and thermalhyperalgesia on days 3, 5, 7, 10 and 14 post-CCI. A significant increase in the levels of Iba (a marker of microgliaactivation) and Bax (a proapoptotic factor) was observed three days after nerve injury. The levels of Iba remained highon day 7. In contrast, there was no difference in glial fibrillary acidic protein (GFAP) contents between sham and CCIanimals. Treatment with the extract significantly attenuated behavioral changes associated with neuropathy. Bax/Bcl-2and Iba1 were decreased in CCI animals treated with the extract.L. citriodora, might have somedegrees of association with the inhibition of microglia activation and apoptotic pathways.The results support the evidence that microglial activation and apoptosis are correlated with painbehaviors. It is suggested that anti-allodynic and anti-hyperalgesic effects, elicited by  Chronic neuropathic pain often accompanies nerveinjury or damage to the nervous system and it can becategorized as central or peripheral pain. Importantmanifestations are spontaneous pain, allodynia (a painfulresponse to innocuous stimulus), and hyperalgesia(increased sensitivity to nociceptive stimulus). As currenttherapies have low efficacy and do not adequatelyalleviate pain , furtherLippia citriodora, Lippia tryphilla or Aloysia tryphilla(verbenaceae), a plant originated from South America,was introduced into Europe at the end of the 17th centuryand is now adapted well to the Mediterranean area. Basedon the region, this plant is variously called, includinglemon verbena (English), Lousia (Arabic), and Behlimooin Persian. The leaves of this plant are used in differenttypes of food and tea due to the lemon-like aroma. Lemonverbena has been widely used in folk medicine for treatmentof various ailments, such as fever, cold, skin infections anddigestive problems. In addition, lemon verbena has beenused based on its anti-spasmodic, anti-pyretic and sedativeeffects  ethanolic extract on the behavioralparameters of rats, undergoing chronic constriction injury(CCI) of neuropathic pain. In addition, the impact ofthis extract was studied in the protein levels of ionizedcalcium binding activated protein 1 1 (Iba1), as a markerof microglia activation, glial fibrillary acidic protein(GFAP), as a marker of astroglia activation, as well as twopro-apoptotic and anti-apoptotic proteins, Bcl2-associatedX (Bax) and B-cell lymphoma (Bcl2) respectively.Anti-inflammatory and anti-nociceptive effects of thisplant have been demonstrated in some studies -16. InrIn this experimental study, Ketamine and xylazine were I.Pinjected at dosages of 64 and 1.6 mg/kg, respectively.Gabapentin was a gift from Tehran Darou Pharm Co.(Iran). Ethanolic extract was obtained in our laboratory. Itwas dissolved in normal saline (0.9%), and administeredI.P, immediately after preparation at the dosages of 50,100 and 200 mg/kg body weight, once a day. Dosageselection was based on the previous studies .AdminisIdentification of the plant was confirmed by botanists inthe Department of Pharmacognosy, Faculty of Pharmacy,Mashhad University of Medical Sciences (Iran). Forpreparing ethanolic extract, the fine powder dried leaves(200 g) were macerated in successive portions of ethanol for 72 hours and the mixture was subsequentlyfiltered and concentrated at 50˚C. The yielded extract wasfreeze-dried and stored at -20˚C .High-performance liquid chromatography-diode arraydetector (HPLC-DAD) was performed on a KNAUERliquid chromatograph system consisting of a quaternarypump . Detection wascarried out using UV-visdiode array detector , and data were processed usingAgilent EZChrom Elite software . Theethanolic extract was subjected to reverse-phase HPLCusing a gradient method of 20-100% methanol (A) inwater as the eluent including 0.05% trifluoroacetic acid.The preparative C18  and flow rate(10 ml/minute) were used. The linear gradient programwas used as follow: 0-10 minute (s), 0-20% methanol; 10-15 minutes, 20-80% methanol; 15-20 minutes, 80-100%methanol; 20-25 minutes, 100% methanol; 25-28 minutes,100-20% methanol; 28-30 minutes, 20% methanol. Thepeaks were monitored at 320 nm and conformed to the Internationally Accepted Principles forLaboratory Animal Use and Care .According to the technique of Bennett and Xie , onesciL. citriodora ethanolic extract , administered at dosage of 1ml/kg for 14 days. On respective days of 3, 7 and 14, thelumbar spinal cord of three animals in each group (exceptgroup 3) was rapidly ejected from the vertebral columnusing a saline-filled syringe, as quickly as possible, andthen separated on dry ice. The reason of examining theL4 and L5 segments was based on the consideration thatthese lumbar segments are the major contributor to thesciatic nerve , ii. CCI animals were treated with normal saline(NS) at a dosage of 1 ml/kg ,iii. Sham group: animals underwent a surgical procedure,without ligature of sciatic nerves, treated with the NS, iv.CCI animals were treated with gabapentin as the referencedrug (100 mg/kg) for 14 days, and v-vii. CCI animalswere treated with ic nerve .rd, 5th, 7th, 10th and 14th postoperative days.Applied tests were as following order: Von Frey 108 test,acetone and finally thermal stimulus.In order to establish baseline values of the nociceptivethresholds, pain related behaviors were evaluated at thenerve-damage hind paw, one day before operation aswell as at the 3A series of calibrated Von Frey filaments , providing forces of 0.6, 1, 2, 4, 6, 8, 10,15, 25 and 60 g (cut off=60 grams), were used to measuremechanical sensitivity of the injured hind paw. Each ratwas placed in a small plastic cage with a metal meshfloor and allowed to acclimate to their surrounding for15 minutes before testing. The filaments were repeatedlyapplied perpendicular to the mid-plantar surface of thehind paw in an ascending stiffness order, until it bent.Each Von Frey filament was applied five times. Whenrats showed at least three brisk withdrawal responses toa filament, the bending force of the filament was defined as the paw withdrawal threshold (PWT). The hind pawwas tested with at least 30 seconds intervals betweenconsecutive stimuli on the injured hind paw .Cold allodynia was assessed using the acetone dropapplication. Animals were placed in the plexiglas boxesand allowed to acclimatize for about 15 minutes. Acetonetest was performed by touching a single bubble of acetoneto the mid plantar surface of each injured hind paw fromthe tip of a 1 ml syringe. A positive response was recorded,if the animal withdraws the paw following application.For each measurement, the paw was sampled five timesand a mean PWF was calculated. It was elapsed at least 3minutes between each test .A plantar (Hargreaves) analgesic meter   was usedAfter detection of pain threshold on the respectivedays, three rats from each group were anesthetized andsacrificed by decapitation. For the protein extraction,the lumbosacral spinal cord segment of animals wasextracted, stored respectively in liquid nitrogen and -80˚Cuntil usage.3VO4), 10 mMβ-glycerophosphate, 0.2% W/V sodium deoxycholate,1 mM phenylmethylsulfonyl fluoride (PMSF), andcomplete protease inhibitor cocktail .The homogenate was sonicated on ice with three timesof 10-seconds bursts at high intensity with a 10-secondscooling period between each burst and then centrifuged(10000 g) for 10 minutes at 4˚C. Protein concentrationwas determined by Bradford assay kit  andadjusted , 2 mM EDTA, 2 mM EGTA, 10 mMNaF, 1 mM sodium orthovanadate (Naadjusted .Each adjusted sample was mixed 1:1 v:v with 2x sodiumdodecyl sulfate (SDS) blue buffer, boiled, aliquoted andkept at -80˚C. Samples were loaded (50 mg of protein/lane),electrophoresed in a 12% SDS-polyacrylamide gel (SDSPAGE)and blotted to a polyvinylidene fluoride (PVDF)membrane . The membranes were incubatedovernight at 4˚C with mouse monoclonal anti-GFAP , mouse monoclonal anti-Iba-1 , rabbit polyclonalanti-Bax , rabbitpolyclonal anti-Bcl2 and rabbit polyclonal anti-β-actin antibodies . After washing three times withTBST, membranes were incubated with rabbit horseradishperoxidase-conjugate anti-rabbit IgG  or anti-mouse IgG . Enhanced chemiluminescence was used to visualize the peroxidase-coated bands. Theintegral optical density (IOD) of each band was measuredusing Alliance 4.7 gel-doc (UK), and UVtec software (UK)was used for densitometric analysis of the bands. Proteinlevels were normalized against intensity of β-actin protein,as control.All behavioral data are presented as mean ± SEM.Statistical evaluation for behavioral data was made bytwo-ways ANOVA with repeated measure, followed byBonferronis’ post hoc test for multiple comparisons. OnewayANOVA followed by Tukeys’ was used for westernblot results. Differences were considered statisticallysignificant, if P<0.05. Statistical analysis was done byGraphpad Prism 6.01 .L.citriodora showed major peaks at the wavelength of 320nm, for the retention times (minute) of 7.43, 12.11, 12.51,14.2, 15.48 and 15.83 .All of the rats had undergone sciatic nerve CCI, exhibiteddecreased thresholds of mechanical allodynia in theaffected hind paw, 3 days after surgery, as compared tosham animals . Low dose of ethanolic extract(50 mg/kg) did not modify CCI-induced mechanicalhypersensitivity to Von Frey filaments. Mechanicalallodynia-like behavior was significantly attenuated in100 and 200 mg/kg of ethanolic extract, compared tocontrol group on 3-14 days post-operation (P<0.001).However, gabapentin at the administrated dose (100mg/kg) was not able to efficiently alleviate mechanicalallodynia after nerve injury .By 3 days post-CCI, cold allodynia was developed inthe ipsilateral paw CCI-NS animals by elevating pawwithdrawal frequencies, as compared to sham animals. Low dosage of ethanolic extract (50 mg/kg) did not modify CCI-induced cold allodynia. Coldhypersensitivity was reduced in treated animals with100 and 200 mg/kg ethanolic extract, on days 3-14 postoperation,as that observed with gabapentin (100 mg/kg)as the reference drug .Three days after CCI, ipsilateral paws exhibitedpronounced thermal hyperalgesia for 14 days after theinjury, compared to the sham animals . As did L. citriodoraethanolic extract significantly decreased Bax and increasedBcl-2 protein levels on day 3. As a result, the ratio of Bax:Bcl-2 was attenuated in L. citriodora-CCI-animals . Bcl-2protein content was increased on day 7 in NS-CCI animals after the injury.The expression of spinal cord Bax protein was increasedin NS-CCI animals, 3 days after injury (P<0.01), while noobvious change was found in the level of Bcl-2 protein ascompared to the sham animals. Hence a significant increasein the Bax: Bcl-2 protein was detected at this time . Treatment of rats with 100 and 200 mg/kg  animals. AlthougWestern blotting results revealed no significant changein the GFAP levels on days 3  and 7 (FL. citriodora ethanolicextract (100 and 200 mg kg) suppressed the activation ofmicroglia 3 and 7 days after surgery, as compared to CCIanimals receiving NS (P<0.05 for 100 and 200 mg/kg).On day 3 after CCI, the level of Iba, a marker of microgliaactivation, was increased in the spinal cord of CCI-NS rats, ascompared to sham group P<0.01, . While, L. citriodora ethanolicextract was unable to increase the pain thresholdin sham animals, indicating that it has no per seanalgesic activity. A period of 14 days intraperitonealethanolic extract of L. citriodora (100 and 200 mg/kg) in neuropathic pain rats reduced mechanicalallodynia, cold allodynia and thermal hyperalgesia,in comparison with NS-CCI animals which wascomparable to reference drug, gabapentin. However,the attenuation of hypersensitivity to innocuousmechanical stimulus was greater than what wasobserved with gabapentin.Our data demonstrated that Iba, a marker of spinal microglia activation, waselevated in the spinal cord of animals on days 3 and7, while it was declined by day 14, following theCCI. The time course of microglial activation wasconsistent with previous studies . GrowingActivation of these cells is characterized bymorphological modifications and enhancedexpression of markers, such as OX-42 and Iba-1 ,28. ConIba was increased 4 and 21 days after L4-L5spinal cord injury in three strains of rat, no significant upregulationor difference in GFAP expression was observedin two strains of rat on day 4. Although, a significant GFAPup-regulation was detected 21 days after nerve injury inonly Wistar rat.However, studies on the activation of GFAP afternerve injury are controversial and require furtherinvestigations. There are many studies claiming thatastroglia are activated after microglia activation indifferent kinds of peripheral or central nerve injuries, 34. TheL. citriodora (100and 200 mg/kg) for 14 days was capable to normalize theincreased Bax/Bcl-2 ratio observed in the CCI animals, atthis time. Bax/Bcl-2 ratio was declined thereafter on days7 and 14, while no difference was detected among NSCCI,sham and neuropathic animals treated with extractsin these days. Our data in the present study supports theevidence that development of neuropathic pain might beassociated with the activation of apoptosis process, inthe CCI of sciatic nerve  and pro-inflammatory cytokines (TNF-αand IL-1β) are released upon the activation of microglia.All of these factors are implicated in pain facilitation andhence, development and maintenance of chronic pain ,41. HPLL.citriodora and its bioactive ingredients have beendemonstrated in various investigations. In a DPPH assayon the blood of rats, L. citriodora increased anti-oxidantenzymes activities, including catalase (CAT), glutathioneperoxidase (GPx) and glutathione reductase (GRed). Incontrast, it decreased myeloperoxidase (MPO) activity,as a marker of inflammation. The phenylpropanoidsverbascoside and isoverbascoside, as well as theirmetabolites were found to be the main constituentsresponsible for the anti-oxidant effects of this plant (in vitro model of Parkinsons’ disease, simultaneoustreatment with verbascoside, markedly attenuatedmethyl-4-phenylpyridinium ion induced apoptotic death,oxidative stress and the activation of caspase-3 (Anti-oxidant and anti-inflammatory effects of is plant .In an iaspase-3 .L. citriodora extracts decreasedthe reactive oxygen metabolites, thiobarbituric acidreactivesubstances and markedly increased the serumcontent of vitamins A and E (L. citriodora (Verbascoside was more active than ibuprofen in theacetic acid-induced writhing test and showed similareffects in the tail flick test . Studies A and E . Followi A and E . Verbasc A and E . In a re A and E . Althougtriodora .Fourteen days after administration of L. citriodoraethanolic extract, tolerance was not determined and theanti-hyperalgesic and anti-allodynic effects were stillevident up to the end of study. It is therefore unlikelythat relief of the nociceptive responses, induced byverbascoside, could be attributed to opioid receptors.Isacchi et al.  also repL. citriodora could be a good option as an adjunctive therapy in the treatment of neuropathic pain. Antinociceptiveeffects of ethanolic extract of L. citriodoracan be attributed, at least in part, to the anti-apoptotic andmicroglia inhibiting activities.


The advent of big data in biology offers opportunities while poses challenges to derive biological insights. For maize, a large amount of publicly available transcriptome datasets have been generated but a comprehensive analysis is lacking.ra1, ba1, gt1, tb1, tga1) that control plant architecture and kernel structure, while multiple other modules relate to diverse agronomic traits. Contained within these modules are transcription factors acting as known or potential expression regulators for the genes within the same modules, suggesting them as candidate regulators for related biological processes. A comparison with an established Arabidopsis network revealed conserved gene association patterns for specific modules involved in cell organization, nutrients uptake & utilization, and metabolism. The analysis also identified significant divergences between the two species for modules that orchestrate developmental pathways.We constructed a maize gene co-expression network based on the graphical Gaussian model, using massive RNA-seq data. The network, containing 20,269 genes, assembles into 964 gene modules that function in a variety of plant processes, such as cell organization, the development of inflorescences, ligules and kernels, the uptake and utilization of nutrients (e.g. nitrogen and phosphate), the metabolism of benzoxazionids, oxylipins, flavonoids, and wax, and the response to stresses. Among them, the inflorescences development module is enriched with domestication genes  contains supplementary material, which is available to authorized users. Advances over the past two decades have generated numerous transcriptome datasets. Increasingly, ever more complete transcriptome data can be merged and integrated with gene and genome structures. They provide unbiased snapshots of gene expression dynamics within organisms under various conditions. While cell-, organ-, or condition-specific expression profiles abound, it remains a key challenge to deduce the underlying gene regulatory circuits that control and give rise to the observed gene expression dynamics. To address this, gene network analysis has emerged as a tool that can filter and refine the analysis of large gene expression datasets. Such gene networks consist of genes (nodes) and connections (edges) between genes that represent co-expression dynamics or association patterns underlying the expression data. According to a ‘guilt-by-association’ paradigm, connected genes may have similar functions, be part of the same complex or pathway, or participate in the same signaling circuits . Gene neDifferent from random networks, gene networks generally identify gene modules categorizing and tracing groups of highly inter-connected genes that share similar expression patterns and, often, are recognizable by their relationship to a function in a particular biological process. Each module can be viewed as a unit in control of one or several biological functions. As such, modules identify and bring together segments of a biological system. Gene network analysis has been used to detect gene modules associated with, for example, human diseases or plant seed germination, and to study the transcriptome landscapes and gene module organization in yeasts, plants, and animals –10.Gene networks derived from microarray data have been described for plant species such as Arabidopsis, maize, and rice , 11–13. Yet another way to approach co-expression network analysis uses the graphical Gaussian model (GGM), which utilizes partial correlation (Pcor) to identify association relationships between genes –17. SignMore recently, a large collection of RNA-Seq based maize gene expression datasets have been generated by different groups and deposited in the public domain. Among these datasets are a gene expression atlas for 79 maize tissues , 20, as Here, we constructed a maize GGM gene co-expression network using expression data from 787 RNA-Seq runs deposited in the NCBI SRA database. 964 gene modules were identified from this network, highlighting functions in various cell organization, development, nutrients, metabolism, and stress responses pathways. As examples, we describe in detail modules involved in the development of inflorescences, ligules, and kernels, the uptake and utilization of nitrogen and phosphate, the metabolism of benzoxazionids, oxylipins, flavonoids and wax, and the response to heat stress, endoplasmic reticulum (ER) stress, and fungal infections. These modules provide a general picture for the relevant biological processes and identified both known genes as well as potential, so far unconnected, candidate genes for future functional studies. Importantly, many of these modules contain transcription factor genes that act as potential gene expression regulator for the genes within the same modules. In addition, the maize network has been compared to a previous published Arabidopsis network , demonstA maize gene co-expression network based on GGM was constructed using maize RNA-Seq transcriptome datasets deposited in the NCBI Sequence Read Archive (SRA) database. The publicly available raw data files (.sra) were downloaded, mapped against the maize reference genome AGP_v3.22), and processed into gene expression values. After removing files with mapping rates <70%, 787 RNA-Seq runs from 36 different studies  of the network is 0.209, while the expected C of a random network of the same size is only 0.0006 [C measures a network’s potential modularity [C of the maize GGM network indicates high characteristics of modularity. Indeed, a network clustering procedure using the MCL program [i. Cell organization; ii. Development; iii, Nutrient uptake & utilization; iv. Metabolism, including primary & secondary metabolism; and v. Stress responses . The clustering coefficient  and ba1 , two TF genes with opposite roles in tassels development [ra1 and ba1 have increased and zero number of tassel branches respectively. A number of genes for the development of ears and tassels were included as well: ra3 (ramosa3), sk1 (silkless ears1), spi1 (sparse inflorescence1), tb1 (teosinte branched1), gt1 (grassy tillers1), and bd1 (branched silkless1), [tga1 (teosinte glume architecture1), a gene conferring the naked kernel phenotype in maize [GRMZM2G022606 and GRMZM2G026556, two genes homologous to BOP2 in Arabidopsis. In Arabidopsis, BOP1, BOP2, and PUCHI (a bd1 homologue) redundantly promote floral meristem fate [ereb161 and nactf114, homologous to Arabidopsis genes ANT and CUC3 that regulate ovule and embryonic apical meristem formation, respectively [ra1, ba1, gt1, tb1, and tga1, have been subjected to selection during maize domestication and were designated as domestication genes [Maize plant architecture is a major yield-determining trait. Our gene network identified three particularly relevant modules. ure Fig. : Fig. S2elopment , 45. Maikless1), –51. Alsoin maize . Uncharatem fate , raisingectively , 55. In on genes , 56. IntModule #48 , encoding a SBP TF that acts as a mater regulator of ligule development [lg1 mutations in maize erases ligules and renders leaves more upright compared to wild type leaves [sbp3 and sbp28, are also included within the module, although their functions remain uncharacterized. Other potential development regulators included myb44, a homologue of Arabidopsis LOF1 that functions in organ boundary specification [GRMZM2G480687, encoding a MEMBRANE-ASSOCIATED KINASE REGULATOR (MARK) proteins, and GRMZM2G145909, homologous to an Arabidopsis atypical bHLH gene IBL1. MARKs and atypical bHLH TFs in Arabidopsis participate in developmental processes mediated through brassinosteroids (BR) [Module #210 contains 15 genes, eight of which encode TFs in the KNOX family (Table gn1 (gnarley1), kn1 (knotted1), rs1 (rough sheath1), lg3 (liguleless3), and lg4 (liguleless4), none of which show any expression in the pre-ligule regions of the wild type plants  , 62, whiids (BR) . Module Module #46  [o2 (opaque endosperm2) and a Dof TF gene pbf1 (prolamin-box binding factor1), two master regulators of zein gene expression [ereb167, platz12, nrp1, nactf130, as potential transcription regulators. Module #6 is expressed in both embryo and endosperm tissues at late developmental stages , desiccation tolerance genes (e.g. genes encoding late embryogenesis abundant proteins), and the TF gene vp1 (viviparous1), a master regulator of seed maturation and dormancy  (#11), the embryo-surrounding region (ESR) (#32), and the placento-chalazal region (PC) (#53)  , 68. Notpression , 70. It Other modules were identified that draw attention to the development of other tissues and organs , nitrite reductase (nii2), and glutamine synthetase  [GRMZM2G161459 and GRMZM2G057616, homologues of the Arabidopsis genes NRT1.1 (encoding a nitrate transporter and sensor) and CLC-A (encoding a vacuole nitrate transporter required for high nitrate uptake capacity) [GRMZM2G047474 and GRMZM2G133684, whose homologues in Arabidopsis AT5G39590 (encoding a TLD domain containing protein) and AT5G62720  are also included in an Arabidopsis nitrate-responsive module but they remain uncharacterized. Their appearance in nitrate responsive modules in both species strongly suggests functions in nitrate sensing, uptake and/or utilization. Also conserved are two uncharacterized LOB-type TF genes lbd6 and lbd11, homologous to one Arabidopsis nitrate response regulator gene LBD37 [lbd6 and lbd11 arguably could have similar functions in maize. Additionally, the maize module includes genes encoding G2-like, HB, and C2H2 type TFs, whose functions in nitrate response have not yet been tested.Nutrient use efficiency is a key objective for crop improvement. Modules can be pinpointed from the maize network as functioning in nitrogen, phosphate, iron, and sulfate uptake and utilization. In general, these modules are conserved between maize and Arabidopsis. For example, 3, gln6) . Also coapacity) , 76. Comne LBD37 , indicatModule #36 includes genes involved in the phosphate starvation response   and sulfate (#79) were also identified , bx2, bx3, bx4, bx5, bx6, and bx8 that form a gene cluster in chromosome 4, and bx9 in chromosome 1 [bx genes share any conserved interactions with the Arabidopsis network, consistent with the absence of benzoxazinoids biosynthesis pathway in Arabidopsis. Additionally, the module is enriched with oxylipins biosynthesis genes , such as lox2, lox3, lox5, lox6, and lox10. The inclusion of both benzoxazinoids and oxylipins biosynthesis genes implies this module’s function in defense response.Gene modules were identified for various processes in maize metabolism. In addition to those involved in primary metabolisms, numerous modules functioning in secondary metabolism emerge as well. Some of these modules are unique to maize, while others share considerable similarities between Arabidopsis and maize. Among them, genes in mosome 1 . InteresModule #154  and r1 (colored1), two TF genes controlling anthocyanin biosynthesis [myb105 and wrky33. In Arabidopsis, TTG2, a homolog of wrky33, regulates tannin level in the seed coat [wrky33 remains to be tested. The maize module also includes the gene mrpa3, encoding a tonoplast-localized anthocyanin transporter [mrpa6 and AC206266.3_FG001. In addition to Module #154, involvement in flavonoids biosynthesis is indicated for Module #65 as well.bz1, bz2 . The modynthesis , and twoeed coat , while tnsporter , and twoModule #40  in maize and MYB94 in Arabidopsis, homologous to each other, both identified recently as TF regulators of wax biosynthesis [gl3 (glossy3), another MYB TF as a master regulator of wax production [ereb12, ereb60, and ereb143. Among them, ereb60 shows homology to Arabidopsis WRI1 that regulates the accumulation of fatty acids – precursors for wax biosynthesis [Waxes, deposited on the aerial surface of plants as a water-proof layer, are essential for plants in that they not only significantly limit water loss, but also counteract environmental stresses . Module  and gl3 . Among tnd LACS1 . Anotherynthesis , 88. Theoduction . Other pynthesis . AdditioIn addition to benzoxazinoids, oxylipins, flavonoids, wax, and fatty acids, modules were also identified for the metabolism of suberin (#42), trehalose #61), glucose (#234), glucan (#278), and lignins (#712)  and a co-activator gene MBF1C (GRMZM2G051135), highlighting their overarching importance in regulating heat activated gene expression regulation. The splicing regulator genes SR45a (GRMZM2G073567) and SR30 (GRMZM2G331811) are also included in the list of conserved genes, suggesting alternative splicing could play important roles in the heat shock response in both species. Similarly, Module #95 , consistent with previous reports that infection by certain fungal pathogens upregulates gibberellin related genes in maize [Also identified from the network were modules related to biotic stress responses. For example, amalexin . Althougin maize .Additional defense related modules were identified from the network, for example Module #2 and #47  ranged from 0% to 83% for different modules . Thus, combining the modular arrangements revealed by the gene network with detailed leaf segment RNA-Seq datasets provided a coherent and detailed picture of maize leaf development, maturation, and biochemical activities. The results were consistent with the previous report [The maize gene modules identified from our network were used to re-analyze a previously published dataset that had measured transcriptomes from 15 segments of maize leaves, from the base to the tip, representing pronounced developmental gradients . Owing ts report , 96, andWe report on the construction of a maize GGM gene co-expression network that includes 20,269 genes based on large-scale RNA-Seq transcriptome data. The resulting gene network was then analyzed and clustered via the MCL clustering algorithm . Althougra1/3 in Module #16, o2, pbf1 in Module #46, bx1/2/3/4/5/6/8 in Module #80, and pl1, r1 in Module #154 [ra1, ba1, gt1, tga1, and tb1 that have been shown by mutants analysis to regulate maize inflorescences architecture, and these 5 genes are designated as domestication genes since they were subjected to selections in the history of maize domestication as they control desirable traits [The modules identified in our network analysis covered many aspects of maize biology. Compared to a previously published maize transcriptional network that contained 49 modules , our module #154 , 81, 82.e traits , 52, 56,o2 and pbf1, are contained within this modules as well. In yet another example, the master regulator TF of seed maturation, vp1, is included within the seed maturation Module #6. The ligule-related Module #48 contains the master regulator gene lg1, encoding a SPL TF. The genes within this module are enriched a SPL binding motif, indicating they are targets of Lg1. Similarly, the heat-induced Module #10 contains 6 heat shock TF genes hsft7/8/12/20/24, the ER-stress response Module #95 includes a putative maser regulator bzip60, and the phosphate starvation response Module #38 possesses three putative master regulator glk4, glk5, glk7. Significantly, many identified modules also included genes encoding unknown and uncharacterized TFs that may represent novel gene expression regulators. The modular structures, as revealed by our network, provide an expedient and edifying way to identify putative TF regulators for various maize pathways.An important feature of our network is that transcription factors and their target genes are often contained within the same module, suggesting shared expression characteristics. One example is the endosperm development Module #46, which is enriched with zein storage protein genes. The regulatory genes for zein biosynthesis, TF The maize gene network also enables cross-species comparison between maize and Arabidopsis. The comparison revealed an unexpected degree of insight into different degrees of conservation in different pathways. Not surprisingly, cell organization modules showed the highest percentage of conserved genes, indicating the evolutionary stability of such basic cellular pathways. Among development related modules, those involved in generating basic building blocks of the plant body are shown to be conserved as well, i.e. carpels, Casparian strip, epidermis, and cell walls. However, divergence was found for functions in modules related to the overall architecture of the plants, for example the development of inflorescence structures. Such comparison sheds light on pathways that might have been “hot targets” for evolutionary changes. In the near future, with RNA-Seq transcriptome datasets rapidly accumulating, such network comparison analysis can be extended into more plant species to identify steps that highlight and determine plant evolutionary trajectories. As shown here for maize and Arabidopsis, conversation and similarity in modular comparisons will assist in pinpointing key regulators in various modules that can then be analyzed in detailed studies.In conclusion, the maize GGM network presented here - in juxtaposition with a corresponding Arabidopsis network  - sheds http://hannonlab.cshl.edu/fastx_toolkit/). Sequence quality was examined using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and low quality read was filtered by FASTX-toolkit. The remaining reads were then mapped to the maize genome AGP v3.22  using Tophat v2.0.10 [The publicly available maize RNA-Seq transcriptome datasets deposited in the NCBI SRA database were used in the analysis. These datasets were organized by studies. The studies were manually inspected to filter out those focusing on non-coding RNAs or those measuring transcriptome of the same tissues from a large number of maize varieties. Also removed were the studies with less than five RNA-Seq runs or without published articles. As a result, 36 studies were kept and their raw data files (sra) were downloaded. For RNA-Seq data processing, adapter sequences, if present, were removed from raw sequence reads using FASTX-toolkit pipeline version 0.0.13 ( v2.0.10  with def v2.0.10 .2 (FPKM + 1) function, a procedure that significantly reduced the dataset’s mean-variance dependency  were filtered out, resulting in a matrix with 29,316 genes and 787 columns. The matrix was log-transformed –40 via tTo evaluate the effect of log-transformation on the network quality, another gene network was also constructed directly from the gene expression matrix with the original FPKM values without data transformation, designated as non-transformed FPKM network, keeping all other parameters the same as in the log-transformation-based network outlined above. These two networks were then evaluated and compared via the EGAD package in R regarding their capacities to connect maize genes with shared GO terms . The resThus, we considered the higher power of the log-transformation-based network, and only results using the log-transformation-based network, designated as the Maize GGM Network, were further analyzed and discussed.http://bioconductor.org/packages/RBGL/) is used to calculate the clustering coefficient of the maize GGM network. The network was clustered via the MCL clustering algorithm, using these parameters “-I 1.5 -Scheme 7” [ftp://ftp.gramene.org/). The maize genes and their Arabidopsis homologues were further annotated with annotation files from MaizeGDB, TAIR, and PlnTFDB [The R package of RBGL v 1.44.0   was usedAdditional file 1: Table S1.Table S4. Selected gene modules identified from the network. Table S5. The percentage of genes with conserved interactions within each module. (XLSX 45 kb)The RNA-Seq studies used in the analysis. Additional file 2: Figure S1.a The standard deviation of each gene’s non-transformed FPKM expression values across all 787 RNA-seq runs are shown against the rank of genes. Genes are ranked by their mean expression values, from low (left) to high (right). The red line depicts a trend line of standard deviation, which indicates a clear mean-variance dependency. b The standard deviation of each gene’s expression values after log transformation across all RNA-Seq runs against the rank of genes. The mean-variance dependency is greatly reduced. (PDF 5820 kb)Log-transformation reduced the mean-variance dependency of the maize RNA-Seq data. Additional file 3: Table S2.The 123,666 gene pairs used for the maize GGM gene network construction. (TXT 4378 kb)Additional file 4: Table S3.The 964 gene modules identified from the network. Listed are the genes’ names and their module identity, and the results of gene ontology enrichment analysis. (XLSX 3251 kb)Additional file 5: Figure S2.A heatmap showing the tissue-specific gene expression patterns for the inflorescences, ligules, and kernels development related modules. The data source of the SRA studies were labeled in the sample names. Listed are the maize genes names and its symbol in lowercase letters, or, if it has no symbols, the names of its Arabidopsis homologues. (PDF 2927 kb)Additional file 6: Figure S3.A heatmap for modules related to endosperm development. (PDF 2005 kb)Additional file 7: Figure S4.A heatmap for additional modules related to development. (PDF 4276 kb)Additional file 8: Figure S5.Figure S6. A module for heat-shock stress response. Figure S7. A module for ER-stress response. Figure S8. A module for stress response to fungus. Figure S9. Comparison between the non-transformed FPKM network and the log-transformation-based network. Both networks were evaluated via the EGAD package [A module for wax biosynthesis.  package  in R reg


We performed a network meta-analysis to compare the efficacy of 12 single-drug regimens  in the treatment of type 2 diabetes mellitus (T2DM). Fifteen relevant randomized controlled trials (RCTs) were included; direct and indirect evidence from these studies was combined, and weighted mean difference (WMD) and surface under the cumulative ranking curves (SUCRAs) were examined to evaluate the monotherapies. Liraglutide was more effective than Glimepiride, Pioglitazone, Sitaglitin, Exenatide, and Glipizide at reducing glycated hemoglobin (HbA1c) levels. In contrast, Acarbose was less effective than Glibenclamide, Glimepiride, Pioglitazone, Rosiglitazone, Repaglinide, Metformin, and Liraglutide at decreasing HbA1c levels. Reductions in fasting plasma glucose (FPG) levels were similar after all treatments. Rosiglitazone was less effective than Glibenclamide and Repaglinide at reducing total cholesterol (TC) levels. High density lipoprotein (HDL), low density lipoprotein (LDL), and triglyceride levels did not differ after treatment with any of the monotherapies. HbA1c and FPG SUCRA values were highest for Liraglutide, while HbA1c and FPG values were lowest for Acarbose, and TC and LDL values were lowest for Rosiglitazone. These results suggest that Liraglutide may be most effective, and Acarbose least effective, at reducing blood glucose levels, while Glibenclamide, Repaglinide, and Metformin may be most effective, and Rosiglitazone least effective, at reducing lipoidemia, in T2DM patients. Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease associated with hyperglycemia, which can lead to serious vascular complications . Both geVarious single-drug regimens are commonly used to treat T2DM. Metformin, a biguanide derivate, has pleiotropic effects beyond glucose reduction, including improvement of lipid profiles and reduction microvascular and macrovascular complications associated with T2DM . PioglitIn this study, we initially retrieved 1,240 relevant studies, of which 8 were duplicated publications, 201 were letters or reviews, 198 were non-human research, and 268 were not in English and were eliminated. Of the remaining 565 studies, we excluded 178 non-RCT studies, 112 studies unrelated to T2DM, 259 studies that did not use single-drug regimens, and 1 study without sufficient data. Ultimately, 15 eligible RCTs published between 1999 and 2017 that included a total of 3,597 T2DM patients were included in this network meta-analysis  Supplem. More paWe carried out direct paired comparisons of the efficacy of 12 single-drug T2DM treatment regimens. Patients who received Glimepiride had higher FPG and HbA1c levels than those who received Liraglutide , which indicated that Liraglutide was more effective than Glimepiride at lowering blood glucose levels in T2DM patients. Compared to those receiving Acarbose, FPG and HbAlc levels were lower in patients who received Pioglitazone , which demonstrated that the hypoglycemic effects of Acarbose were relatively poor. Patients taking Glibenclamide had lower LDL levels , and those who took Pioglitazone had lower TC and LDL levels , than patients treated with Rosiglitazone, indicating that Rosiglitazone was less effective at decreasing blood lipid levels . . Compared to Acarbose, HbA1c levels were lower in patients treated with Glibenclamide, Glimepiride, Pioglitazone, Rosiglitazone, Repaglinide, Metformin, or Liraglutide , which suggested that Acarbose was relatively ineffective at reducing blood glucose levels. Compared to Repaglinide, patients who received Glipizide had higher HbA1c levels , indicating that Glipizide was less effective at lowing blood glucose levels. However, all drugs were similarly effective in reducing FPG levels. TC levels were lower in patients treated with Glibenclamide or Repaglinide than in those treated with Rosiglitazone , which indicated that Rosiglitazone was less effective at decreasing blood lipid levels. Finally, all drugs had similar effects on HDL, LDL, and triglyceride levels in T2DM patients . We therefore conducted a sensitivity analysis by removing the Gudipaty et al. (2014) study and performing the statistical analysis, sorting the interventions, and calculations of cumulative probability rankings again. The rankings of the interventions were largely unchanged in the repeated analysis, indicating that the Gudipaty et al. (2004) study did not have a significant impact on our conclusions  studies were randomized controlled trials (RCTs); (2) single-drug regimens  were used to treat T2DM; (3) study subjects were 40- to 80-year old patients diagnosed with T2DM; (4) FPG, HbA1c, TC, high density lipoprotein (HDL), LDL, or triglycerides were measured to evaluate outcome. The exclusion criteria were: (1) patients treated with insulin or who had diabetic complications that required treatment; (2) patients with severe deficiencies in liver and kidney function; (3) patients with cardiovascular disease within 3 months of the study; (4) patients who were pregnant or lactating women; (5) patients with severe hypertension; (6) available experimental data was incomplete; (7) non-RCTs; (8) duplicated publications; (9) conference reports, systematic reviews or summaries; (10) studies published in languages other than English.Two researchers independently extracted data from the included studies using a standardized data collection form. Disputes regarding data extraction were discussed and negotiated by several researchers until a consensus was reached. Cochrane Collaboration's tool was used to assess the risk of bias in the randomized controlled trials . The tooP > 0.05 were analyzed using the consistency model [We performed traditional pairwise meta-analyses for studies that directly compared different treatment arms. Pooled estimates of weighted mean differences (WMD) and 95% confidence intervals (CIs) are reported. Chi-square test and I-square tests were employed to test heterogeneity among the studies . R 3.2.1cy model . To assicy model , 43. Clucy model . All com


The G subunit of Juglans regia (JrVHAG1) was previously identified as a drought tolerance-related gene involved in the ABA (abscisic acid)-signal pathway. Heavy metal stress is becoming a major detriment for plant growth, development, and production. In order to understand the role of JrVHAG1, the potential function mechanism of JrVHAG1 exposed to CdCl2 stress was confirmed in this study.Vacuolar HJrVHAG1 was induced by ABA and increased to 58.89-fold (roots) and 7.38-fold (leaves) and by CdCl2 to 2.65- (roots) and 11.42-fold (leaves) relative to control, respectively. Moreover, when treated simultaneously with ABA and CdCl2 (ABA+CdCl2), JrVHAG1 was up-regulated to 110.13- as well as 165.42-fold relative to control in the roots and leaves, accordingly. Compared to the wild type (WT) Arabidopsis plants, the transgenic plants with overexpression of JrVHAG1  exhibited increased seed germination rate, biomass accumulation, proline content, and activities of superoxide dismutase (SOD) and peroxidase (POD) under ABA, CdCl2, and ABA+CdCl2 treatments. In contrast, the reactive oxygen species (ROS) staining, malondialdehyde (MDA) content, hydrogen dioxide (H2O2) content, as well as electrolyte leakage (EL) rates of transgenic seedlings were all lower than those of WT exposed to ABA, CdCl2 and ABA+CdCl2 stresses. Furthermore, a 1200 bp promoter fragment of JrVHAG1 was isolated by analyzing the genome of J. regia, in which the cis-elements were identified. This JrVHAG1 promoter fragment showed expression activity that was enhanced significantly when subjected to the above treatments. Yeast one-hybrid assay and transient expression analysis demonstrated that JrMYB2 specifically bound to the MYBCORE motif and shared similar expression patterns with JrVHAG1 under ABA, CdCl2 and ABA+CdCl2 stress conditions.Transcription of JrVHAG1 gene functions as a CdCl2 stress response regulator by participating in ABA-signal pathway and MYB transcription regulation network. JrVHAG1 gene is a useful candidate gene for heavy metal stress tolerance in plant molecular breeding.Our results suggested that the The online version of this article (10.1186/s12870-018-1231-7) contains supplementary material, which is available to authorized users. Heavy metals include micronutrients , trace elements , and stress factors  that are toxic to plants . Heavy mAmong the stress related heavy metals, Cu, Zn, Pb, Ni, Se, Cr, and Co were moderately enriched, whereas Sb and cadmium (Cd) were extremely highly enriched in the majority of soil samples, therefore, Cd is regarded as one of the most phytotoxic heavy metals , 5. In sOsPCS2) from Oryza sativa improved Cd and As stress tolerance by mitigating the accumulation of these heavy metals in rice plants . Every treatment was replicated three times and each replicate contained at least 30 seedlings.The t genome , and ampe/html/) . To undep method . Four-wep method  under noJrVHAG1 promoter  were found in the d assays .CAGGG” and inserted into pHis2 (pHis2-MYB-M). Fragments of the JrVHAG1 promoter including the MYBCORE motif (pHis2-MYB-S), excluding the MYBCORE motif (pHis2-MYB-M1), and including the mutated MYBCORE motif (pHis2-MYB-M2) were all cloned into pHis2, respectively  expression  was cloned into prokII vector such that it is placed under the control of a 35S promoter (prokII-JrMYB2) .All of the data were analyzed using the Statistical Package for Social Science (SPSS) . The differences between the transgenic and WT lines were evaluated using Tukey’s multiple comparison test with the significance level set at


Altererythrobacter sp. strain B11 is an aromatic monomer-degrading bacterium newly isolated from sediment under the seabed off Kashima, Japan, at a depth of 2,100 m. Here, we report the complete nucleotide sequence of the genome of strain B11. Altererythrobacter is one of the genera within Alphaproteobacteria proposed by Kwon et al. , at a depth of 2,100 m. The strain B11 can degrade aromatic monomers, such as p-coumaric acid, ferulic acid, and 4-hydroxybenzoic acid, which are components of various plant cell walls  according to the manufacturer’s protocol. Whole-genome sequencing of strain B11 was performed by means of both Pacific Biosciences RS II  and IlluThe genome of strain B11 is composed of a single circular chromosome , with a mean G+C content of 65.4%. We identified 3,645 protein-coding sequences (CDSs), 51 tRNAs, and 6 rRNAs by means of the MetaGeneAnnotator , tRNAscaAltererythrobacter species in marine environments.We predicted overall metabolic and physiological functions of strain B11 using the metabolic and physiological potential evaluator (MAPLE) with bidirectional best-hit matches , 16. In AP018498.This whole-genome shotgun project has been deposited in DDBJ/ENA/GenBank under the accession no.