Thr models -9. To st The lymphocyte antigen-6 (Ly6) complex, a group of alloantigens, was first discovered in mice approximately 40 years ago on lymphocytes , 4. Ly6 Here we evaluate the importance and significance of novel Ly6 family in cancer prognosis and treatment using publically available datasets of gene expression micro array analysis coupled with clinical outcome information. To study the status of Ly6D, Ly6E, Ly6H and Ly6K mRNAs in human normal and cancer tissues in one-hundred and thirty gene expression omnibus (GEO) dataset using Oncomine (Invitrogen) or Georgetown Database of Cancer (G-DOC). The expression status of Ly6D, Ly6E, Ly6H and Ly6K in caner tissue was correlated with patient outcome using KM plotter and PROGgeneV2 platform.To examine the status of Ly6D in human cancer, we used Oncomine or G-DOC to analyze gene expression omnibus (GEO) datasets. The data summarized in Table These results show that Ly6D expression was significantly increased in bladder, brain and CNS, breast, head and neck, gastric, lung, ovarian, pancreatic, colorectal and kidney cancer than their counterpart normal tissues.The data summarized in Table These results show that Ly6D expression was significantly increased in subtypes of bladder, brain and CNS, breast, pancreatic, gastric, cervical, colorectal, esophageal and kidney cancer.Table p=0.012, n= number of patient, HR=hazard ratio), post progression free survival , relapse free survival  shown by KM plotter and five-year relapse free survival  shown by PROGgeneV2  and overall survival  shown by PROGgeneV2  or with restriction of lung adenocarcinoma , first progression free survival with no restriction  or with restriction of lung adenocarcinoma  and post progression free survival with restriction of lung adenocarcinoma , by KM plotter and five-year relapse free survival with restriction of lung adenocarcinoma  shown by PROGgeneV2  shown by KM plotter  shown by KM plotter  datasets. The data in Table Table 3These results show that Ly6E expression was significantly increased in bladder, breast, esophageal, gastric, pancreatic, cervical, colorectal, prostate, lung, head and neck, ovarian, kidney, melanoma, embryonic cancer than their counterpart normal tissues.The data in Table These results show that Ly6E expression was significantly increased in subtypes of bladder, brain, esophageal, pancreatic and breast cancer.Table High Ly6E mRNA expression in neuroblastoma was significantly correlated with five-year recurrence free survival  in Asgharzadeh  study. Hp=0.0026, n= number of patient, HR=hazard ratio), shown by PROGgeneV2 (p=0.022) or without restriction , five-year relapse free survival with restriction of grade 1 breast cancer , grade 2 breast cancer , or without restriction , five-year distant metastasis free survival , five-year post progression free survival , shown by KM plotter. High Ly6E mRNA expression in breast cancer was significantly correlated with poor five-year overall survival with restriction of estrogen receptor positive breast cancer , with restriction of progesterone receptor positive breast cancer  or without restriction , and five-year distance metastasis free survival  shown by PROGgeneV2  shown by KM plotter  and poor five-year relapse free survival with restriction of stage IIb cancer  or with restriction of stage 1b cancer  shown by PROGgeneV2  shown by KM plotter  or without any restriction  shown by PROGgeneV2  datasets. As shown in Table These results show that Ly6H expression was significantly increased in brain and CNS, esophageal, breast, kidney, head and neck, lung and ovarian cancer than their counterpart normal tissues.As shown in Table These results show that Ly6H expression was significantly increased in subtypes of brain and CNS, breast, gastric, kidney, pancreatic, cervical, and colorectal cancer.Table p=0.0326, n= number of patient, HR=hazard ratio) shown by PROGgeneV2 , five-year post progression free survival  and five-year overall survival  shown by KM plotter  shown by KM plotter and five-year overall survival  shown by PROGgeneV2  and five-year overall survival  shown by KM plotter  shown by PROGgeneV2 (p=0.002); five year overall survival with restriction of stage IIIb cancer  and with restriction of stage IIb cancer  shown by PROGgeneV2 (p=0.0008) shown by PROGgeneV2 (p=0.059) shown by PROGgeneV2  are up regulated in cancerous tissue than normal tissue, the Increased expression of these genes are heterogeneous among difference subtypes of multiple cancer and that the high expression of these genes is significantly associated with poor outcome. Recently Butte et al described that the gene expression data of tumor mass can be influenced by infiltration by immune cells and non-cancerous normal cells . These cThe mouse and human Ly6 family have a conserved LU domain Figure . The LU The Ly6K and Ly6E proteins are implicated as cancer vaccine targets and drug conjugated antibody therapy, respectively -9 suggeswww.oncomine.org) [www.cbioportal.org) [http://www.elsevier.com/solutions/pathway-studio) [kmplot.com/analysis) [http://kmplot.com/analysis/) [http://www.abren.net/PrognoScan/) [ONCOMINE (ine.org) , 152 wastal.org) , 154 for-studio)  was usednalysis)  was usedalysis/)  and PrognoScan/)  were useProtein sequences of the LY6K, LY6E, LY6D, LY6H genes from both human and mouse plus the Ly6A gene from mouse were obtained from UniProtKB . In addi


Suppressing cardiac and respiratory motion artifacts are major challenges in cardiac MRI. The conventional methods of ECG and diaphragm navigator gating require tedious setup, reduce the imaging efficiency significantly, are susceptible to drifts in heart rate or respiratory pattern, and can be unreliable at higher field strengths . In this3, matrix = (384)3, and a 3D radial trajectory with 2D golden means [For data acquisition, we used a contrast-enhanced, ungated spoiled gradient-echo sequence with the following sequence parameters: TR/TE = 5.5/3.0 ms, flip angle = 15 degrees, water excitation hard pulse, FOV = 400 mm)00 mm3, mFigure We have demonstrated a fully self-gated 4D whole-heart imaging technique with high isotropic spatial resolution and near 100% imaging efficiency through respiratory motion correction and retrospective cardiac gating. The golden 3D radial trajectory allows one to freely trade between image quality and temporal resolution, and one may determine the precise quiescent period retrospectively, which can span one or more reconstructed cardiac phases, and select the appropriate subset of data to reconstruct a high-resolution motion-free image. Future investigations are warranted to further optimize the reconstruction and sequence parameters, as well as to compare the proposed method with the conventional prospectively gated protocol.NIH Grant Numbers: HL38698, EB002623.


Mytilus galloprovincialis and Crassostrea gigas. In detail, we have identified and phylogenetically compared eight evolutionary conserved proteins: DROSHA, DGCR8, EXP5, RAN, DICER TARBP2, AGO and PIWI. In mussels, we recognized several other proteins participating in the miRNA biogenesis or in the subsequent RNA silencing. According to digital expression analysis, these genes display low and not inducible expression levels in adult mussels and oysters whereas they are considerably expressed during development. As miRNAs play an important role also in the antiviral responses, knowledge on their production and regulative effects can shed light on essential molecular processes and provide new hints for disease prevention in bivalves.Small non-coding RNAs include powerful regulators of gene expression, transposon mobility and virus activity. Among the various categories, mature microRNAs (miRNAs) guide the translational repression and decay of several targeted mRNAs. The biogenesis of miRNAs depends on few gene products, essentially conserved from basal to higher metazoans, whose protein domains allow specific interactions with dsRNA. Here, we report the identification of key genes responsible of the miRNA biogenesis in 32 bivalves, with particular attention to the aquaculture species  Different types of non-coding RNAs (ncRNAs) have gained attention for their powerful regulatory action on eukaryotic genes and other genetic elements . The proClamydomonas and Ectocarpus spp.) while they are apparently absent in protists  are the microprocessor’s core proteins which allow interactions with the DDX5 helicase, the RNA binding protein Lin-28 and hnRNP A1, among other elements (Exportin5 (XPO5) by interaction with the small GTPase RAN; then, they are further processed by the RISC loading complex, composed by the endoribonuclease DICER, the RNA binding protein TARBP2 and Argonaute proteins  and the Maelstrom protein (MAEL)  .Cnidaria  technologies, miRNAs have been deeply explored in non-model organisms, including bacteria , plants Cnidaria , Platyhelminthes  and inselminthes . Regardilminthes , miRNA flminthes  and one lminthes . A generrochozoa  to identM. galloprovincialis (Mg) and C. gigas (Cg) as well as in other bivalve and non-bivalve species . A Mg reference transcriptome was produced using 18,788 ESTs of mixed tissues previously obtained by Sanger sequencing (PRJNA88481) (SRP033481) (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) discarding the reads with PHRED quality below 20 and presenting more than two ambiguous nucleotides. De-novo assembly was performed with Trinity, release 2013-08-14 (spp. (Cg plus other 29 species) were retrieved from the SRA archive and assembled as described above  and the00297895 ) were requencing  and 453 NA88481) , and froP033481) . The qua13-08-14 , setting13-08-14 . Transcrntissima .To investigate the presence of eight key proteins of miRNA biogenesis , we downloaded their predictive HMM from PFAM v.27 (listed in blastn) in order to recover the related gene structures. Positive hits having e-value lower than 10−20 were extracted and assembled on the corresponding transcript, used as backbone. RNA-seq read mappings with adapted parameters  allowed us to ascertain the correct gene assembly. Homolog gene structures were retrieved by interrogating genomic browsers, like Metazome v.3  and Ensembl Metazoa v.29  or by local blastn against the downloaded genomes .We used the transcript sequences of DROSHA, DGCR8, XPO5, DICER and TARBP2 as blast queries against all Mg genomic contigs (http://tree.bio.ed.ac.uk/software/figtree/).The inferred protein sequences were aligned using MUSCLE, release 2014-05-29 . SubsequTo analyze the expression of the selected genes in Cg and Mg RNA datasets, we retrieved all available RNA-seq samples from the NCBI SRA archive. For Cg, we analyzed 123 Illumina RNA-seq samples related to adult tissues or developmental stages. For Mg, we analyzed 13 RNA samples from gills (1), digestive gland (6), haemocytes (2), mantle (2) and muscle (2). Overall, we included in the expression analysis 2,271 and 453M reads for Cg and Mg, respectively . The triMytilus galloprovincialis transcripts involved in the miRNA biogenesis by systematic searches of diagnostic domains  and two PIWI-like proteins (PAZ and PIWI domains). We considered the above mentioned gene products as the key complement of the miRNA biogenesis.Among the possible interacting proteins, we identified MgGW182, a transcript encoding a protein shorter than the human counterparts but holding all the features considered significant for its interaction with AGOs and the CCR4-NOT complex. In fact, MgGW182 possesses 19 N-terminal GW stretches, followed by one UBA domain, a Q-rich region (M domain) and a C-terminal RNA recognition motif (RRM domain). Moreover, we recognized a C-terminal conserved site known as PAM2 , expecteTaking advantage of mussel WGS data  we invesC. gigas, L. gigantea and A. californica. Since the C. gigas genome includes annotations only for the cds regions, we exploited full-length transcripts obtained from a locally assembled oyster transcriptome to expand the genome annotations in this species. In particular, we updated the annotation of CgDGCR8 and CgDICER and we added new annotations for CgPIWI-1  and CgTARBP2 .To identify the miRNA biogenesis complements in marine mollusks, we used homologous genes retrieved from the genomes of spp. do not have at present a sequenced genome, we used publicly available RNA-seq data to build 29 specie-specific transcriptome assemblies and retrieve the homologous sequences of interest. After domain searching, we carefully considered the high number of positive hits to retain only proteins including all the expected protein features. Thus, we retrieved 132 complete hits from marine mollusks: 10 DROSHAs, 9 DGCR8s, 14 XPO5s, 34 RANs, 7 DICERs, 13 TARBP2s and 45 Argonaute-like proteins, the latter classified in 13 AGO and 32 PIWI proteins by phylogenetic analysis  being the most far-related  clustering with Arthropoda and more distantly Platyhelmintes and Rotifera hits. The Chordata sequences clustered as a separate group ; four genes in plants like A. thaliana and P. trichocarpa and one gene in Ctenophora, Rotifera, Cephalopoda Mollusca and Chordata. Moreover, the presence of DICER was reported in some Protozoa and fungi (The finding of putative DICER sequences in Ctenophora volution . Also plnd fungi . Phylogend fungi . Likewisnd fungi . We backspp. (with the exception of 15 proteins in A. pisum) and three or four different proteins in bivalve spp. Also, basal Metazoa possess Argonaute-like sequences: four in the genomes of Ctenophora and Cnidaria spp., one in the Placozoa T. adhaerens and two in A. queenslandica. The case of C. elegans is remarkable since it holds several Argonaute gene families and at least 24 proteins  as normalizer housekeeping gene to compare the expression level of the different genes in each sample.We used the 13 Mg and 124 Cg RNA-seq samples to evaluate the expression levels of miRNA biogenesis genes in different tissues and conditions. Based on total mapped reads, we computed TPM values and we used elongation factor 1 α, except for DDX5, RAN and CNOT9). Mantle and muscle appeared the most responsive tissues whereas haemolymph was the least responsive one. In particular, the genes that we considered as the core components of miRNA biogenesis were expressed at levels below 0.5% of El1α , with the exception of the high levels of CgPIWI-1 levels in male and female gonads  and two PIWI transcripts from the egg to trocophora  and developmental stages (39) . In adulocophora . In the spp., with particular attention to M. galloprovincialis and C. gigas. To the best of our knowledge, we report for the first time the presence of a complete miRNA biogenesis pathway in M. galloprovincialis, the full-length transcript sequences of DICER, DGCR8, XPO5, RAN, DROSHA, TARBP2, three Argonaute genes and the identification of many other components that are candidate miRNA complement-interacting proteins such as MgGW182. By using local transcriptome assemblies, we identified these genes also in many other marine bivalves. The general low expression levels of these transcripts in the adult tissues of both M. galloprovincialis and C. gigas, and the considerable gene size, have probably prevented a previous identification of full-length sequences in not-well-covered bivalve transcriptomes. In fact, we obtained complete transcript sequences only from sequenced genomes or highly-covered transcriptomes whereas in other transcriptome assemblies we retrieved only few complete sequences. Overall, we have analyzed 523 miRNA complement sequences, 145 of them belonging to marine mollusks and displaying a consistent sequence clustering .Small RNAs are important regulators of the gene expression, as recognized in various model and non-model organisms , includiC. gigas and A. californica genomes coding for four proteins (2 AGO and 2 PIWI proteins) whereas M. galloprovincialis and L. gigantea possess three proteins (1 AGO and 2 PIWIs). We also highlighted the over-expression of the miRNA biogenesis genes during the first phases of the oyster development. A genome protection mechanism based on piRNA expression during early developmental stages is well known in mammals , species name and order, origin of the sequenced sample, sequencing platform, millions of reads, number of assembled contig and number of predicted proteins are reported.Click here for additional data file.10.7717/peerj.1763/supp-2File S2Click here for additional data file.10.7717/peerj.1763/supp-3File S3A, CNOT1; B, CNOT2; C, CNOT3; D, CNOT6; E, CNOT7; F, CNOT9; G, CNOT10; H, DCP1, I, DCP2; J, PAN2; K, PAN3; L, eIF4G; M, PABP; N, PRMT5; O, DDX5; P, DDX6; Q, DDX20; R, DDX42; S, TUDOR-11 and T, MAEL. Domain organization of each protein is shown, green bars represent coiled-coil regions and purple bars disordered regions. Length is expressed as amino acid scale above the figure.Click here for additional data file.10.7717/peerj.1763/supp-4File S4Click here for additional data file.10.7717/peerj.1763/supp-5File S5Click here for additional data file.10.7717/peerj.1763/supp-6File S6Digital Expression values of the miRNA biogenesis genes measured by RNA-seq analysis for Mg and Cg (expressed as percentage of El1a).Click here for additional data file.


Arthrobacter is one of the most prevalent genera of nicotine-degrading bacteria; however, studies of nicotine degradation in Arthrobacter species remain at the plasmid level (plasmid pAO1). Here, we report the bioinformatic analysis of a nicotine-degrading Arthrobacter aurescens M2012083, and show that the moeB and mogA genes that are essential for nicotine degradation in Arthrobacter are absent from plasmid pAO1. Homologues of all the nicotine degradation-related genes of plasmid pAO1 were found to be located on a 68,622-bp DNA segment (nic segment-1) in the M2012083 genome, showing 98.1% nucleotide acid sequence identity to the 69,252-bp nic segment of plasmid pAO1. However, the rest sequence of plasmid pAO1 other than the nic segment shows no significant similarity to the genome sequence of strain M2012083. Taken together, our data suggest that the nicotine degradation-related genes of strain M2012083 are located on the chromosome or a plasmid other than pAO1. Based on the genomic sequence comparison of strain M2012083 and six other Arthrobacter strains, we have identified 17 σ70 transcription factors reported to be involved in stress responses and 109 genes involved in environmental adaptability of strain M2012083. These results reveal the molecular basis of nicotine degradation and survival capacities of Arthrobacter species. FB24 (FB24), Arthrobacter chlorophenolicus A6 (A6), and Arthrobacter phenanthrenivorans Sphe3 (Sphe3), and a food-processing strain, Arthrobacter arilaitensis Re117 (Re117), were reported. Strains FB24, A6 and Sphe3 were isolated by their ability to degrade dimethylbenzene, 4-chlorophenol, and phenanthrene, respectively. Genome analyses revealed many metabolic and stress-related genes in these environmental strains, which reflected their pollutant-degrading abilities and niche specializations. Strain Re117 was isolated from the surface of Reblochon cheese; its genetic adaptation to the habitat via genes such as those involved in iron acquisition, salt tolerance, and catabolism of fatty acids, amino acids, and lactic acid, which are major carbon substrates present at the cheese surface, was also revealed by genome sequence analysis323435The complete genome sequences of four other N-heterocyclic pollutant and Arthrobacter is one of the most prevalent genera of nicotine-degrading bacteria, studies of nicotine degradation by Arthrobacter species remain at the plasmid level, while the complete genome of another prevalent bacterium, Pseudomonas putida S16, has been sequenced and extensively studied373839404142Arthrobacter nicotinovorans (formerly classified as Arthrobacter oxydans) was sequenced, and almost all the genes involved in nicotine degradation by the bacterium were shown to locate on it373838Arthrobacter strains, which is very important to their application potential, is still unknown. Therefore, it is necessary to obtain and analyze the genome sequence of a nicotine-degrading Arthrobacter strain in order to uncover the molecular basis of its survival capacity.Although nicotine is a typical A. nicotinovorans were possibly obtained via plasmid pAO1 or via a precursor plasmid from the chromosome of a strain that was probably related to Rhodococcus opacus or another Arthrobacter species. However, Arthrobacter species harboring nicotine-degrading genes on the chromosome have not yet been reportedMany catabolic plasmids are thought to take part in the spread of catabolic traits by horizontal gene transfer between bacteria4647Arthrobacter sp. M2012083 (M2012083), from tobacco waste, and obtained and published its genome sequence, but without further analysisArthrobacter aurescen by physiological and biochemical identification, 16S rDNA phylogenetic analysis, and genomic analysis; we compared the genomic sequence of strain M2012083 with the six available Arthrobacter complete genomes and the sequence of nicotine-degrading plasmid pAO1, in order to provide a better understanding of the genetic basis of the nicotine-degradation and survival capacities of this genus.Recently, we isolated a new nicotine-degrading strain, Arthrobacter, but were also detected in members of other genera such as Brevibacterium and Rhodococcushttp://blast.ncbi.nlm.nih.gov/Blast.cgi) indicated that the 16S rDNA sequence of strain M2012083 exhibited more than 99% nucleotide acid sequence identities with that of TC1 and Rue61a. We selected 55 16S rDNA sequences from different strains in order to construct a phylogenetic tree by using MEAG5 analysis with the neighbor-joining method51A.aurescens and A.nitroguajacolicus . Strain M2012083 is gram-positive, aerobic, and asporous. Transmission electron microscopic observation revealed that the cells of strain M2012083 were long rods in their lag phase (6 h) and became coccus-shaped during the log phase (36 h) in nicotine-containing medium. These morphological characteristics are representative of the genus acolicus . Furtheracolicus . Based oArthrobacter genomes are summarized in Arthrobacter strains and to provide a quick overview of the conserved genomic regions18Arthrobacter genomes, 68–79% of protein-coding genes were functionally categorized based on the Clusters of Orthologous Groups (COGs) databaseThe general features of the seven subject Arthrobacter strains, which have no homologous genes in the other six strains, are listed in Orthologous relationship analysis was performed using the OrthoMCL methodIn order to reveal the nicotine-degradation mechanism of strain M2012083, its genome was searched for all reported bacterial nicotine degradation-related genes and the protein-coding genes of plasmid pAO1, using the tBLASTp program3738414243565758Arthrobacter strains except Re117, which contained only 74 ORFs  was highest in the genomes of the six environmental Arthrobacter strains, FB24, TC1, A6, Sphe3, Rue61a, and M2012083, while genes related to replication, recombination, and repair (COG L) were the most numerous in the Re117 genome, in which the percentage of COG G proteins (6.5%) was much lower than that in the other subject genomes (>11.8%); this is probably because strain Re117 locates on the surface of cheese, which is rich in proteins rather than carbohydrates. Furthermore, the numbers of genes categorized as COG C, K, R, S, and T in the Re117 genome are also much lower than those in other subject genomes, which indicates that surviving on the surface of cheese requires less functions related to energy transformation, transcription, or signal transduction. The COGs have highly similar distributions among the six environmental Arthrobacter strains, except for COGs L and N. Genes with COG L annotations, representing replication, recombination, and repair, are more numerous in Sphe3 than in the other five strains. There are 15 genes annotated as COG N in A6, which is far more than that in other strains (only 0 or 1), suggesting that all the subject Arthrobacter strains lacked cell motility, except strain A6. Genes annotated as COG B, the group related to chromatin structure and dynamics, were not found in any of the seven subject genomes , 6-hydroxy-L-nicotine oxidase (6hlno), 6-hydroxy-D-nicotine oxidase (6hdno), and ketone dehydrogenase subunits , and those involved in γ-N-methylaminobutyrate catabolism (mgaba), MoCo biosynthesis (moco), assembly and quality control of the (αβγ)2 holoenzyme complexes (aqc), compound transport (perm1 and perm2), and gene regulation , were located on a 68,622-bp DNA segment (nic segment-1) of contig 058 in the M2012083 genome, which showed 98.1% nucleotide acid sequence identity to the 69,252-bp nic segment of pAO1  and considerably more than that in strain Re117, which only encodes six  and betaine aldehyde dehydrogenase gene (betB), and a gene cluster that encodes the four subunits of sarcosine oxidase, are present in all seven Arthrobacter genomes. A second copy of betA was identified in FB24, TC1, and A6, while a second copy of betB is found only in FB24 and A6. Many glycine betaine transporter genes, such as clusters of the glycine betaine ABC transport system permease and opuD genes, were also present in all seven Arthrobacter genomes. Only strains FB24 and A6 contained both the choline binding protein A gene and high-affinity choline uptake protein (BetT) gene; only strains M2012083, TC1, Sphe3, and Rue61a contained the choline binding protein A gene, while only strain Re117 contained the betT gene. Moreover, in strain M2012083, osmotic stress may be reduced by the regulation of water movement, glycerol uptake, and osmoregulated periplasmic glucan synthesis, via aquaporin Z, the glycerol uptake facilitator protein, and opgC, respectively , except that of the food-processing strain, Re117. An ORF encoding SoxR, which is a redox-sensitive activator of the oxidative stress response, is present in M2012083, TC1, Re117, and Rue61a but absent from FB24, A6, and Sphe3 , while only one was found in the food-processing Arthrobacter strain, Re117. Every subject genome contained an ORF encoding cold-shock protein CspC, except the genome of Rue61a. In addition, all the Arthrobacter strains contained a dnaK operon , an hspR gene encoding a transcriptional repressor of the dnaK operon, a heat-inducible transcription repressor hrcA gene, and several other genes encoding heat-shock response proteins. Interestingly, an additional one or two dnaJ, dnak, and grpE genes were identified in the M2012083 genome, but these genes were not located in an operon; similar data were observed in strain Rue61a  and a derivative thereofL-asparateArthrobacter genomes . A phylogenetic tree was constructed by the neighbor-joining method using MEGA5 analysis51The 16S rDNA of strain M2012083 was amplified by PCR with the universal primer pair of 27F (5′-AGAGTTTGATCCTGGCTCA-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) from extracted genomic DNA. The PCR product was purified and sequenced, then analyzed by homology alignment using the BLAST search program . Whole-genomes of strain M2012083 were sequenced by the Chinese National Human Genome Center . The Velvet program was used to assemble the pair-end reads The National Institutes of Allergy and Infectious Diseases/Pathosystems Resource Integration Center (PATRIC) and RAST were used for genome comparisons and metabolic pathway analyses. The tBLASTn program was used to search for homologs of plasmid pAO1 protein-coding genes in the M2012083 genome with coding amino acid sequence identity > 30% at an e-value < 1e-5. The MUMmer program was used to generate a synteny plot of plasmid pAO1 versus the M2012083 genome. The OrthoMCL method was used to analyze the orthologous relationships between protein-coding genes in different genomes. Genes in different genomes with coding amino acid sequence identity > 50% at an e-value < 1e-5 were identified as orthologous genes. We used the CRISPRFinder program in CRISPR's web server to identify clustered regularly interspaced short palindromic repeats in the CRISPR/Cas RNA-guided nuclease systemArthrobacter aurescens M2012083 has been deposited in NCBI database under the accession number AKKK00000000. The accession numbers of the subject genomes are as following: Arthrobacter sp. FB24 chromosome, NC_008541; Arthrobacter sp. FB24 plasmid 1, NC_008537; Arthrobacter sp. FB24 plasmid 2, NC_008538; Arthrobacter sp. FB24 plasmid 3, NC_008539; Arthrobacter aurescens TC1 chromosome, NC_008711; Arthrobacter aurescens TC1 plasmid TC1, NC_008712; Arthrobacter aurescens TC1 plasmid TC2, NC_008713; Arthrobacter chlorophenolicus A6 chromosome, NC_011886; Arthrobacter chlorophenolicus A6 plasmid pACHL01, NC_011879; Arthrobacter chlorophenolicus A6 plasmid pACHL02, NC_011881; Arthrobacter arilaitensis Re117 chromosome, NC_014550; Arthrobacter arilaitensis Re117 plasmid pRE117-1, NC_014549; Arthrobacter arilaitensis Re117 plasmid pRE117-2, NC_014548; Arthrobacter phenanthrenivorans Shpe3 chromosome, NC_015145; Arthrobacter phenanthrenivorans Shpe3 plasmid pASPHE301, NC_015146; Arthrobacter phenanthrenivorans Shpe3 plasmid pASPHE302, NC_015147; Arthrobacter nitroguajacolicus Rue61a chromosome, NC_018531; Arthrobacter nitroguajacolicus Rue61a plasmid pAL1, NC_009453; Arthrobacter nitroguajacolicus Rue61a plasmid p232, NC_018532; Arthrobacter nicotinovorans plasmid pAO1, NC_021229.The genome sequences of P.X., Y.Y. and H.T. conceived and designed the project and experiments. Y.Y., S.F. and H.T. performed the computational and bioinformatic analysis. Y.Y., H.T. and P.X. wrote the paper. All authors reviewed the paper.Supplementary informationDataset 1Dataset 2Dataset 3Dataset 4


In vivo study showed that administration of garcinol alone  significantly suppressed the growth of the tumor, and this effect was further increased by cisplatin. Both the markers of proliferation index (Ki-67) and microvessel density (CD31) were downregulated in tumor tissues by the combination of cisplatin and garcinol. The pharmacokinetic results of garcinol indicated that good systemic exposure was achievable after i.p. administration of garcinol at 0.5 mg/kg and 2 mg/kg with mean peak concentration (Cmax) of 1825.4 and 6635.7 nM in the mouse serum, respectively. Overall, our results suggest that garcinol can indeed potentiate the effects of cisplatin by negative regulation of various inflammatory and proliferative biomarkers.Platinum compounds such as cisplatin and carboplatin are frequently used as the first-line chemotherapy for the treatment of the head and neck squamous cell carcinoma (HNSCC). In the present study, we investigated whether garcinol, a polyisoprenylated benzophenone can chemosensitize HNSCC to cisplatin. We found that garcinol inhibited the viability of a panel of diverse HNSCC cell lines, enhanced the apoptotic effect of cisplatin, suppressed constitutive as well as cisplatin-induced NF-κB activation, and downregulated the expression of various oncogenic gene products .  Lysates were then spun at 13,300 rpm for 10 min to remove insoluble material and resolved on a 10% SDS gel. After electrophoresis, the proteins were electrotransferred to a nitrocellulose membrane (Bio-Rad), blocked with Blocking One , and probed with antibodies of interest overnight at 4ºC. The blot was washed with TBS with 0.1% Tween-20, exposed to HRP-conjugated secondary antibodies for 1 h, and finally examined by chemiluminescence using Western Bright Sirius HRP substrate (Advansta).Vehicle or drug-treated whole-cell extracts were lysed in lysis buffer t-test and one way analysis of variance, (ANOVA). A p value of less than 0.05 was considered statistically significant. AbbreviationsHNSCC, head and neck squamous cell carcinoma; NF-κB, nuclear factor kappa B; COX-2, cyclooxygenase-2; MMP-9, matrix metalloproteinase 9 ICAM-1, intercellular adhesion molecule 1, FAK, focal adhesion kinase HAT, histone acetyltransferase, STAT3, signal transducer and activator of transcription 3, IκBα, inhibitor of NF-κB alpha, VEGF, vascular endothelial growth factor, IKKβ, IκB kinase betaStatistical analysis was performed by Student's


Actinobacillus pleuropneumoniae isolates, is described. The new primers eliminate an aberrant serovar 3-indicative amplicon found in some serovar 6 clinical isolates. Furthermore, we have developed a new multiplex PCR for the detection of serovars 1 to 3, 5 to 8, 10, and 12 along with apxIV, thus extending the utility of this diagnostic PCR to cover a broader range of isolates.An improved multiplex PCR, using redesigned primers targeting the serovar 3 capsule locus, which differentiates serovars 3, 6, and 8  Actinobacillus pleuropneumoniae is a major cause of morbidity and mortality due to respiratory disease in pigs and is responsible for substantial economic losses worldwide  a doublA. pleuropneumoniae strains , the United Kingdom (n = 154) (including 9 serovar 6 isolates that previously tested positive for serovars 3 and 6), Cyprus (n = 6), Switzerland (n = 6), and China (n = 2). All the isolates were previously typed serologically and/or by capsule-specific PCR. In addition, 31 strains of other porcine-associated bacterial species were tested as negative controls These included Actinobacillus suis (n = 5), Actinobacillus minor (n = 3), Actinobacillus porcitonsillarum (n = 3), Actinobacillus porcinus (n = 3), Actinobacillus indolicus (n = 3), Haemophilus parasuis (n = 5), Pasteurella multocida (n = 3), Bordetella bronchiseptica (n = 2), Mycoplasma hyopneumoniae (n = 3), and Streptococcus suis P1/7 (n = 1).In this study, we used 15 reference A. pleuropneumoniae serovar 6 United Kingdom clinical isolate MIDG2472, which shows the double-banding pattern using the 3-6-8 multiplex PCR previously described .The primers used in this study are detailed in KJ685492 and KJ685493, respectively.The DNA sequences of the capsule loci of strains MIDG2472 and 405 have been deposited in GenBank under accession numbers cps3A (APJL_1614) to cpxD (APJL_1615), and the serovar 8 primers (AP8NF and AP8NR) were designed to amplify a 1,106-bp fragment from cps8A to cps8B, as indicated in cpsD) gene  were derived from a previously published PCR , 12. TheA APJL_16 to cpxD sD) gene . A compasD) gene  eliminatADOD00000000 , ADOE00000000 , CP000687 , ADOF00000000 (M62 serovar 4), CP000569 , ADOG00000000 , CP001091 , ADOI00000000 , ADOJ00000000 , ADOK00000000 , ADOL00000000 , and ADOM00000000 . The details of the primer pairs that were derived for use in the 9-serovar multiplex PCR are provided in A. pleuropneumoniae reference strains . We recommend that where an isolate is strongly suspected to be A. pleuropneumoniae and there is a lack of either apxIV or a serovar-specific amplicon in our 9-serovar PCR, researchers retest the isolate with alternate A. pleuropneumoniae-specific primers such as those indicated above. If the isolate is confirmed as A. pleuropneumoniae, it is likely that its serovar is not covered by our multiplex PCR. While other multiplex PCRs have been reported for all but serovar 10, these have typically involved coverage of three serovars  or are comparatively rare in the United Kingdom. Serovar-specific sequences were derived from whole-genome sequences: GenBank accession numbers  strains . As expeIVA-TSP2 , and eacA. pleuropneumoniae, which were derived from our collection and have been assigned at the species level by conventional biochemical testing. None of these strains produced any amplicons with the new 9-serovar multiplex PCR.The specificity of the multiplex PCR was further tested using 31 isolates representing porcine-associated bacterial species other than A. pleuropneumoniae serovars 1 to 3, 5 to 8, 10, and 12. With the use of our well-characterized collection of A. pleuropneumoniae isolates and other porcine-associated bacterial species, the new PCR was 100% sensitive and specific for serotyping. In regard to the United Kingdom, the PCR covers all of the serovars  that were previously reported  of strains that failed to produce the predicted 418-bp amplicon. In those strains, an apxIV band was obtained with alternate primers. Although we did not investigate further, the new multiplex PCR could be adapted to detect relevant serotypes found in different countries by excluding or including capsule-specific primers for other serovars as required.The inclusion of


Phyllostomus hastatus probably retaining the ancestral karyotype for the subfamily. Since chromosomal rearrangements occur rarely in the genome and have great value as phylogenetic markers and in taxonomic characterization, we analyzed three species: Lophostoma silvicola (LSI), Phyllostomus discolor (PDI) and Tonatia saurophila (TSA), representing the tribe Phyllostomini, collected in the Amazon region, by classic and molecular cytogenetic techniques in order to reconstruct the phylogenetic relationships within this tribe. LSA has a karyotype of 2n=34 and FN=60, PDI has 2n=32 and FN=60 and TSA has 2n=16 and FN=20. Comparative analysis using G-banding and chromosome painting show that the karyotypic complement of TSA is highly rearranged relative to LSI and PHA, while LSI, PHA and PDI have similar karyotypes, differing by only three chromosome pairs. Nearly all chromosomes of PDI and PHA were conserved in toto, except for chromosome 15 that was changed by a pericentric inversion. A strongly supported phylogeny (bootstrap=100 and Bremer=10 steps), confirms the monophyly of Phyllostomini. In agreement with molecular topologies, TSA was in the basal position, while PHA and LSI formed sister taxa. A few ancestral syntenies are conserved without rearrangements and most associations are autapomorphic traits for Tonatia or plesiomorphic for the three genera analyzed here. The karyotype of TSA is highly derived in relation to that of other phyllostomid bats, differing from the supposed ancestral karyotype of Phyllostomidae by multiple rearrangements. Phylogenies based on chromosomal data are independent evidence for the monophyly of tribe Phyllostomini as determined by molecular topologies and provide additional support for the paraphyly of the genus Tonatia by the exclusion of the genus Lophostoma.The subfamily Phyllostominae comprises taxa with a variety of feeding strategies. From the cytogenetic point of view, Phyllostominae shows different rates of chromosomal evolution between genera, with  Subfamily Phyllostominae Gray, 1825, includes taxa that have diversified feed strategies, including carnivorous, strictly insectivorous and a combination of frugivorous and insectivorous ,4. The dPhyllostominae bats represent a taxonomically controversial group. Although phylogenetic analyses of morphology , alloenzLophostoma, Mimon, Phylloderma, Phyllostomus, Tonatia (Phyllostomini), Macrophyllum, Trachops (Macrophyllini), Chrotopterus and Vampyrum (Vampyrini). This arrangement was recently supported by Hoffmann et al. [Macrotus, Micronycteris, Lampronycteris, Lonchorhina, Trinycteris and Glyphonycteris which were classified within Phyllostominae according to Koopman [Baker et al.  proposedn et al. . The gen Koopman , McKenna Koopman , Simmons Koopman , Wettere Koopman , and Jon Koopman , were reTonatia using morphological characters. The monophyly of Tonatia based on morphological characters was questioned by Arnold et al. [T. bidens and other species of Tonatia were equally as great as between T. bidens and Phyllostomus. In the most recent review, Lee et al. [Tonatia, except T. bidens and T. saurophila, to the genus Lophostoma. This same arrangement was found by Porter et al. [Within Phyllostomini, Goodwin  and Genod et al.  and by Hd et al. , becausee et al.  classifir et al.  in theirTonatia [Lophostoma silvicola [Phyllostomus, Mimon, Phylloderma, and in some species of Lophostoma, to the species with a high degree of karyotypic evolution as found in the karyotypes of Tonatia bidens, T. saurophila and Lophostoma schulzi [Tonatia, although the homology of chromosome arms between Tonatia and other genera cannot suggest certainty based solely on G-banding.From the chromosomal point of view, previous studies in Phyllostomini showed a high variation in diploid number with 2n = 16 for Tonatia ,23, and ilvicola , 24–25.  schulzi –10,26. B schulzi  proposedTonatia [In this regard, chromosome painting has contributed to the elucidation of chromosomal homologies between species phylogenetically distant –32, or bTonatia .Phyllostomus hastatus and Carollia brevicauda [Dhyphylla eucaudata, Diaemus youngi, Desmodus rotundus (Desmodontinae) [Artibeus obscurus, Uroderma bilobatum, U. magnirostrum (Stenodermatinae) [Micronycteris hirsuta (Micronycterinae) [Tonatia saurophila, Lophostoma silvicola and Phyllostomus discolor from the Amazon rainforest (Brazil), by cross-species chromosome painting using chromosome-specific probes from Carollia brevicauda and Phyllostomus hastatus. We used all Desmodontinae bats as the phylogenetic outgroup [Until now, seven species in three phyllostomid subfamilies were studied by cross-species chromosome painting using probes of evicauda : Dhyphylontinae) , Artibeumatinae) , Micronyterinae) . Here, woutgroup , and comoutgroup . Our aimTonatia saurophila have 2n = 16 and FN = 20, with three pairs being bi-armed and four one-armed chromosomes pairs; the X is a medium-sized submetacentric and the Y a small acrocentric  in the distal region of the short arm of pair 6  revealed 32 homologous segments on the Tonatia saurophila (TSA) genome , showed only one fluorescent signal in the chromosomes of Tonatia, corresponding to segments of TSA-5 (PHA-11 and 15), TSA-6, and whole TSA-X. PHA-5 also hybridized to only one chromosome, the pair 6 of TSA, but in early metaphase with much distended chromosomes, presented two hybridization signals, separated by a region without homology. No other PHA or CBR probe hybridized in this region.ZOO-FISH analyses using whole chromosome probes of ) genome . Four paTonatia: PHA-2 (TSA-1 and 2), PHA-3 (TSA-1 and 4), PHA-4 (TSA-1 and 4), PHA-7 (TSA-1 and 3), PHA-8 (TSA-3 and 5), PHA-9 (TSA-1 and 5), PHA-10 (TSA-2 and 7), PHA-12 (TSA-1 and 2). PHA-13 yielded three hybridization signals in TSA-1, 5 and 7, while PHA-1 showed three signals, but hybridized to two distinct chromosomes: TSA-4 and 7. PHA-6 showed four hybridization signals, corresponding to four chromosomes on TSA-1, 2, 3 and 5.Eight PHA probes gave two fluorescent signals, with each probe marking two distinct chromosomes in Carollia brevicauda (CBR) whole chromosome probes revealed 31 homologous segments on the Tonatia saurophila (TSA) genome (Tonatia chromosomes: CBR-8 (TSA-1 and 5) and CBR-5 (TSA-4 and 7), while CBR-4 probes also hybridized to two chromosomes of Tonatia: TSA-3 and 7, but showed three hybridization signals.Hybridization of ) genome . Three pCBR-6 hybridized to chromosomes TSA-1, 2 and 5, and showed one fluorescent signal on each. CBR-3 gave four signals on four distinct chromosomes of TSA-2, 3, 4 and 7. CBR-2 showed five hybridization signals, but on just four chromosomes on TSA: 1, 2, 4 and 5. CBR-1 hybridized to chromosomes TSA-1, 2, 4 and 6, but showed five markings, because in TSA-6 it shows two hybridization signals, in the same way as in PHA-5. CBR-Y2 showed four signals to four chromosomes of TSA-1, 2, 3 and 5 .Lophostoma silvicola has a karyotype of 2n = 34 and FN = 60, including 28 bi-armed and 4 one-armed chromosomes; the X is metacentric and the Y acrocentric  in the distal portion of the long arm of chromosome 15 and in the short arm of pair 16 (ocentric . CH is l pair 16 .Phyllostomus hastatus (PHA) probes revealed 17 homologous segments on the Lophostoma silvicola (LSI) genome. Most PHA probes correspond to whole chromosomes in karyotype of LSI and only PHA-13 showed two signals, hybridizing fully to LSI-13 and to the short arm of chromosome LSI-15 (Comparative painting with e LSI-15 .Carollia brevicauda (CBR) whole chromosome probes revealed 26 homologous segments on the Lophostoma silvicola (LSI) genome. The number of homologous segments from CBR chromosomes in the karyotype of LSI extended from one to five. Four CBR probes hybridized to whole LSI chromosomes, with only one fluorescent signal in each: CBR-7 (LSI-11), CBR-8 (LSI-13), CBR-9 (LSI-14) and CBR-X.Hybridization of Three paints of CBR marking three different chromosomes on LSI, each gave two fluorescent signals: CBR-4 (LSI-1 and 8), CBR-5 (LSI-4 and 15) and CBR-6 LSI-12 and 16). CBR-3 paint hybridized to three distinct chromosomes on LSI: 1, 7 and 10. CBR-Y2 showed three hybridization signals, but hybridized to just two chromosomes of LSI-2 and 6. CBR-1 showed four signals on chromosomes of LSI-3, 5, 6, 7. CBR-2 showed five signals, but hybridized to four chromosomes of LSI-2, 4, 8 and 9 . Due to the highly conserved karyotype of PDI, when compared to PHA, we have only hybridized two whole chromosome paints corresponding to chromosomes PHA-14 and 15 which we presume to be involved in rearrangements, the latter corresponding to the acrocentric pair with a NOR inverted in PDI . Tonatia was in a basal position within Phyllostomini (bootstrap = 100 and Bremer = 10 steps), followed by a branch grouping Lophostoma and Phyllostomus (bootstrap = 88 and Bremer = 3 steps), and a group formed by P. hastatus and P. discolor that are sister taxa (bootstrap = 70 and Bremer = 1 step). Desmodontinae bats were used as outgroups and were grouped together with a polytomy (bootstrap = 63 and Bremer = 1)  .Tonatia saurophila is similar to those described for specimens from Central America with 2n = 16, FN = 20 [Lophostoma silvicola with 2n = 34, FN = 60 analyzed here is similar to those described by Gardner [L. silvicola despite its wide geographical distribution. The karyotype of Phyllostomus discolor is similar to those described for Central American specimens [The karyotype of  FN = 20 , 25, 42. Gardner  and Bake Gardner , for spepecimens , Southeapecimens –47.T. saurophila is so derived that no chromosomal arms proposed on the ancestral karyotype of phyllostomid could be identified by classical cytogenetics. Here, we identified chromosomal synteny using chromosome painting associated with G-banding. Some chromosomal segments previously suggest as plesiomorphic for Phyllostomidae [Tonatia genome as PHA-14 (CBR-9), PHA-11 (CBR-7) and PHA-15 . This result demonstrates the usefulness of chromosome painting for the identification of chromosomal homologies when classical cytogenetics is non-informative.According to Patton and Baker  and Bakestomidae –41, wereTonatia [Lophostoma silvicola, Phyllostomus hastatus, and P. discolor. PHA has 2n = 32 and FN = 58, PDI also has 2n = 32 but FN = 60, while LSI has 2n = 34 and FN = 60 and differs from PHA only by three chromosome pairs . PDI and PHA have all chromosomes without rearrangements, except for a pericentric inversion in chromosome 15 [Most genera of phyllostomid bats have highly conserved karyotypes, but there are extensive karyotypic variations among some genera, which make intergeneric comparison by G-banding almost impossible , 46, 48.Tonatia . We founosome 15  Fig 5)..Tonatia Phyllostomus discolor and Mimon crenulatum from a fusion between two pairs of acrocentric chromosomes homologous to Macrotus waterhousii chromosomes, one of them NOR-labeled. This hypothesis is supported by the distal position of the NOR-labeled chromosome shared between Phyllostomus hastatus and Phylloderma stenops (acrocentric form), and between P. discolor and M. crenulatum (metacentric form). So, according to these authors, Phyllostomus discolor presents the primitive form of chromosome 15 for the Phyllostomus genus shared with Mimon. On the other hand, our data show that the metacentric form is autapomorphic in Phyllostomus discolor and the acrocentric form is synapomorphic between Lophostoma silvicola and P. hastatus , are pos et al. .An alternative explanation would be that the two forms of pair 15 are not related, having originated from ancestral chromosomal polymorphisms of Phyllostomini, with the fluctuations in size becoming fixed randomly in several branches (hemiplasy), as previously suggested for Chiroptera, where the presence of the chromosomal synteny HSA 1/6/5 is shared by one family (Pteropodidae) and one subfamily (Megadermatinae). This was formerly interpreted to be homoplasic  but mighPhyllostomus, and with Mimon crenulatum [Lophostoma silvicola and Tonatia saurophila this chromosome is found on a fissioned way, but the fissioned segments were subsequently rearranged, with heterochromatin addition in Lophostoma silvicola. So, in both species this chromosome has a derivate morphology in regard of the ancestral state for the tribe.Despite other chromosomal modifications between the compared species, we observe that chromosome PHA-13 was derived from fission as found in the putative ancestral karyotype of leafed-nose bats , in the enulatum . On the Lophostoma silvicola, Phyllostomus discolor and Tonatia saurophila, and compared the results among the Phyllostomidae species mapped so far: PHA, CBR [We mapped by chromosome painting the karyotype of three species of phyllostomid bats: PHA, CBR , AOB, UBPHA, CBR , DEC, DRPHA, CBR , and MHIPHA, CBR  Fig 5)..LophostoPHA, CBR , , and the genus Phyllostomus clade was the lowest possible (1), but for a branch with a small number of characters parsimony-informative since the karyotypes are almost identical and similar to the primitive Phyllostomidae karyotype , even a low Bremer may be significant [The monophyly of the tribe Phyllostomini was supported by a high value of Bremer (10 steps), indicating strong robustness of the analysis . Howevernificant , since tnificant .Tonatia is 16.2 MYA, while Tonatia species arose 12.1 MYA, and the divergence of Lophostoma from the remainder of the Phyllostomini must have been at least 12–13 MYA [Phyllostomus and Phylloderma, and when radically reorganized karyotypes (karyotypic megaevolution) occurred in Tonatia. In Lophostoma there are species that have both types of karyotype evolution.Analysis among genera of Phyllostominae shows that these lineages are relatively old, arising in the Mid-Miocene, and divergences among tribes happened in the Early Miocene. The node that gave rise to the genus 2–13 MYA . This waTonatia species group are similar morphologically to Lophostoma silvicola and distinguished only by width of the lower incisors, analyses of karyotypic and molecular data indicate that they are highly diverged from other species of Lophostoma [Phyllostomus and Tonatia, or Lophostoma and Tonatia were inferred by molecular data, as well as the basal position of Tonatia to Phyllostomini [Tonatia for the Phyllostomini tribe and the relationship of sister-taxon between Lophostoma and Phyllostomus, due the one-armed chromosome 15 shared between these two taxa , Lophostoma silvicola  and Phyllostomus discolor , from states of Pará, Amazonas and Mato Grosso, Brazil. Chromosomal preparations were obtained by direct extraction from bone marrow after Colchicine treatment following Baker et al. [Three species were analyzed cytogenetically: r et al.  and fibrr et al. . Cell lir et al. , LSI accr et al.  and PHA r et al. . JCP hasConventional staining was used for diploid (2n) and fundamental numbers (FN) determination. G-banding followed two distinct methods: trypsin treatment  and saliProchilodus argenteus [P. hastatus (PHA) and C. brevicauda (CBR) [FISH with digoxigenin labeled telomeric probes  were performed according to the manufacturer’s protocol and 18S rDNA probes from rgenteus  were labda (CBR)  were labChromosome painting was performed as previously described , 63. BriPhyllostomus hastatus karyotype retained most of the segments supposed to be ancestral to family Phyllostomidae [Previous results indicated that stomidae , 41. Forstomidae . An exhastomidae , as outgstomidae . All chastomidae  and Dobistomidae  Click here for additional data file.


Rhizoma coptidis, has been manifested with notable anticancer activities. This study aims to investigate the hormetic effect of berberine and its influence on the anticancer activities of chemotherapeutic agents. Our results demonstrated that berberine at low dose range (1.25 ~ 5 μM) promoted cell proliferation to 112% ~170% of the untreated control in various cancer cells, while berberine at high dose rage (10 ~ 80 μM) inhibited cell proliferation. Further, we observed that co-treatment with low dose berberine could significantly attenuate the anticancer activity of chemotherapeutic agents, including fluorouracil (5-FU), camptothecin (CPT), and paclitaxel (TAX). The hormetic effect and thereby the attenuated anticancer activity of chemotherapeutic drugs by berberine may attributable to the activated protective stress response in cancer cells triggered by berberine, as evidenced by up-regulated MAPK/ERK1/2 and PI3K/AKT signaling pathways. These results provided important information to understand the potential side effects of hormesis, and suggested cautious application of natural compounds and relevant herbs in adjuvant treatment of cancer.Hormesis is a phenomenon of biphasic dose response characterized by exhibiting stimulatory or beneficial effects at low doses and inhibitory or toxic effects at high doses. Increasing numbers of chemicals of various types have been shown to induce apparent hormetic effect on cancer cells. However, the underlying significance and mechanisms remain to be elucidated. Berberine, one of the major active components of  Hormesis is defined as a process in which exposure to a low dose of a chemical agent or environmental factor that is damaging at higher doses induces an adaptive beneficial effect on the cell or organism , 2. In bRhizoma coptidis is a highly valued traditional Chinese medicine which has been widely applied in complementary and alternative medicines in China, Korea, India, Japan, and other Asian countries, especially for the treatment of dysentery, cancer, diabetes mellitus, and eczema, mostly used in formulas [Rhizoma coptidis, which is an isoquinoline alkaloid with abundant pharmacological activities [formulas . Berberitivities . Berberitivities , 17. Mortivities , 19 or rtivities . The encIn this study, we investigated whether berberine at relative low doses stimulates the growth of cancer cells, i.e. hormetic effect, and whether this effect attenuates the anticancer activities of chemotherapeutic agents. The underlying molecular mechanisms of hormesis induced by berberine were also investigated.Berberine hydrochloride (BER), 5-Fluorouracil (5-FU) camptothecin (CPT), and Paclitaxel (TAX) were purchased from Sigma-Aldrich . WST-1 cell proliferation and cytotoxicity assay kit, PD98059, and LY294002 were purchased from Beyotime . RPMI 1640 medium, Dulbecco's modified Eagle's medium (DMEM), penicillin-streptomycin, trypsin, fetal bovine serum (FBS), and phosphate buffered saline (PBS) were obtained from Gibco . Antibodies against phosphor-ERK, ERK, phosphor-AKT, AKT and β-actin were purchased from Cell Signaling Technology, Inc. .2. The medium was changed every other day.B16-F10 murine melanoma cell line was purchased from Cell Bank of Type Culture Collection of Chinese Academy of Sciences . Human breast cancer cell lines MDA-MB-231, MDA-MB-468, and MCF-7, and human colon cancer cell line LS-174 cells were obtained from American Type Culture Collection . According to ATCC guideline, cells were cultured in basic medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in a humidified atmosphere of 5% CO3 cells per well in 96-well plate. After 16~20 h incubation, the cells were treated with a series of concentrations of berberine for 24, 48, or 72 h at 37°C. WST-1 reagent (10 μl) was added to each well at 2 h before the endpoint of incubation. The cells were further incubated for 2 h at 37°C, then the absorbance was measured by a SpectraMax® M5 microplate reader  with a test wavelength at 450 nm and a reference wavelength at 690 nm. Inhibitory rate of cell proliferation was calculated comparing with OD value of control group. All assays were performed at least three replicates and repeated three times.Cell proliferation was determined by WST-1 colorimetric assay . Cancer After treatment, cells were washed twice with PBS and lysed with RIPA lysis buffer containing 1% phenylmethylsulfonyl fluoride (PMSF) and 1% protease inhibitor cocktail . The concentration of protein samples was determined using a BCA protein assay kit . Equivalent amounts of proteins from each group were separated using SDS-PAGE electrophoresis, followed by transferring onto an immun-blot PVDF membrane . After being blocked for 1 h in 5% non-fat dried milk with PBST buffer, the membrane was incubated with 1:1000 dilutions of primary antibody at 4°C overnight. β-actin was used as the internal control. PVDF membranes were washed three times in PBST buffer, and followed by incubation with 1: 5000 dilutions of the corresponding second antibody. Specific protein bands were visualized using an ECL advanced Western blotting detection kit . The density of the bands was quantified by Quantity One Software.p < 0.05.Data were expressed as the mean ± standard deviation. One-way ANOVA and Turkey’s Multiple Comparison Test are used in the GraphPad Prism 5.0 software . The value of statistical significance is set at To investigate the hormetic effect of berberine (BER), we tested five cancer cell lines, murine melanoma cell line B16-F10, human breast cancer cell line MDA-MB-231, MDA-MB-468 and MCF-7, and human colon cancer cell line LS-174. Cell viability was determined by WST-1 colorimetric assay. We observed the temporal features of hormetic effect induced by BER on B16-F10 cells. BER at relatively low concentrations significantly stimulated cell growth, while high concentration of BER inhibited cell growth of the tested cancer cell lines. This biphasic dose-response phenomenon was consistent with the typical feature of hormesis . The resWe hypothesized that the hormetic effect of BER could attenuate the anticancer activities of chemotherapeutic agents. To test this hypothesis, low concentrations of BER (2.5–10 μM) combined with camptothecin (CPT), paclitaxel (TAX) or 5 fluorouracil (5-FU) were used to treat B16-F10 cells for 24 h, 48 h and 72 h. As shown in MAPK/ERK1/2 and PI3K/AKT pathways play an important role in cell proliferation and survival , 24, andTo further validate the role of ERK and AKT signaling pathways in hormetic dose response of BER, we tested whether the pathway inhibitors could affect BER-induced cell growth stimulation in B16-F10 cells. As shown in To investigate whether ERK and AKT activation participates in the attenuation of anticancer activity of chemotherapeutic agents by low dose BER, B16-F10 cells were exposed to chemotherapeutic agents alone, or in combination with low dose BER (5 μM) and MEK or PI3K inhibitors. As shown in Rhizoma coptidis, a widely used traditional Chinese medicine, has a wide range of pharmacological activities [Berberine, one of the major active ingredients in tivities , 27. In tivities , 19, sugtivities . More sttivities –11. ThesThe characteristic of hormesis is a biphasic dose-response curve, with stimulatory or beneficial effect at low doses and inhibitory or toxic effect at high doses , 2. The In clinical practice, drug combination is a common-use strategy for cancer treatment, which may increase therapy sensitivity, provide multiple drug targets and avoid potential drug resistant. Berberine also showed synergistic anticancer activity combined with other chemotherapeutic drugs as reported previously , 19. HowHormesis was considered a universal phenomenon in many disciplines of biological and medical sciences, such as toxicology, immunology, aging biology, neuroscience, microbiology, radiology, etc. . It has In summary, our study demonstrated that berberine induced a significant hormetic dose response, in which low dose berberine strongly stimulated the growth of cancer cells, in contrast to its anticancer activity at high doses. Moreover, low dose berberine greatly attenuated or even reversed the in vitro anticancer activity of chemotherapeutic drugs, including CPT, TAX, and 5-FU. These adverse effects were associated with the activation of signaling pathways for cell proliferation/survival and adaptive oxidative stress response in cancer cells, including MAPK/ERK1/2 and PI3K/AKT. As hormetic effect was considered a universal adaptive response to low dose  cytotoxic agents, our study suggested cautious application of natural compounds and relevant herbs, which could cause obvious hormetic response, in adjuvant treatment of cancer.


To the Editor: Dengue is the most prevalent arthropod-borne viral disease in tropical and subtropical countries. Every year, dengue virus (DENV) infections cause more than 50 million cases, 500,000 hospitalizations, and 12,500 deaths worldwide (Flavivirus and is transmitted by Aedes spp. mosquitoes. There are 4 distinct serotypes (DENV-1 to DENV-4), and infection with 1 serotype does not provide long-term, cross-protective immunity against the other 3 serotypes.In New Caledonia, DENV outbreaks have occurred since World War II and have been caused mainly by 1 serotype/genotype introduced from a country to which dengue is hyperendemic. Since 2000, New Caledonia has had recurrent DENV-1 outbreaks  is an emerging mosquito-borne virus that belongs to the genus In New Caledonia, the first cases of ZIKV infection imported from French Polynesia were confirmed at the end of November 2013, and the first autochthonous cases were reported by mid-January 2014. Early in February 2014, the New Caledonia Health Authority declared an outbreak situation. Since February 2014, a total of 1,385 ZIKV laboratory-confirmed cases have been detected, including 35 imported cases . Concomitant with this ZIKV outbreak, circulation of DENV-1 and DENV-3 was reported; >150 cases were biologically confirmed during January–September 2014 , leukopenia  with associated stimulated lymphocytes, and discreet hepatic cytolysis . Serum was analyzed by using real-time reverse transcription PCR (RT-PCR) and was positive for ZIKV as recommended by Lanciotti et al. (Patient 1 was a 14-year-old boy who had fever (39.5°C), headache, arthralgia, asthenia, and myalgia. No hemorrhagic or neurologic findings were reported, and the patient recovered within 3 days. A complete blood count showed mild thrombocytopenia (platelet count 129 × 109 platelets/L) and leukopenia (leukocyte count 2.65 × 109 cells/L). Serum was analyzed by using RT-PCR (Patient 2 was a 38-year-old woman who had fever (40°C), headache, arthralgia, asthenia, myalgia, retroorbital pain, conjunctivitis, diarrhea, nausea, and a diffuse pruritic maculopapular rash. No hemorrhagic or neurologic findings were reported, but signs of illness lasted ≈1 week. The patient had mild thrombocytopenia  from both patients, partial DENV-1 envelope gene for an isolate (KM212960) from patient 2, and partial DENV-3 nonstructural protein 5 gene for an isolate (KM212962) from patient 1. Sequencing was conducted at La Plateforme du Vivant .DENV-1 sequence obtained belonged to genotype I and clustered with DENV-1 sequences isolated in New Caledonia (Serum specimens from both patients were cultured on Vero cells, and supernatants were evaluated by RT-PCR. Each specimen was positive only for DENV, which was probably caused by low viral loads for ZIKV.We report co-infection of 2 patients with DENV and ZIKV; each patient was infected with a different DENV serotype. No synergistic effects of the 2 viral infections were observed because both patients were not hospitalized and recovered after a mild clinical course.During this outbreak, patients in New Caledonia were tested for DENV, chikungunya virus, and ZIKV within the framework of the arboviruses sentinel network, which enabled detection of co-infections. Thus, clinicians should be aware of infections with multiple pathogens in the differential diagnosis of dengue-like illness, especially in patients who returned from tropical regions. This diagnostic procedure could be improved by using multiplex RT-PCR for travelers, given the frequent co-circulation of multiple arboviruses in tropical regions.


Key findings: Vitamin D metabolites do not consistently influence replication or clearance of rhinovirus, respiratory syncytial virus (RSV) or influenza A virus in human respiratory epithelial cell culture, although they do modulate expression and secretion of type 1 interferon, chemokines including CXCL8 and CXCL10 and pro-inflammatory cytokines, such as TNF and IL-6. Future research: More studies are needed to clarify the effects of vitamin D metabolites on respiratory virus-induced expression of cell surface markers mediating viral entry and bacterial adhesion to respiratory epithelial cells.Background: Vitamin D deficiency has been shown to be independently associated with increased risk of viral acute respiratory infection (ARI) in a number of observational studies, and meta-analysis of clinical trials of vitamin D supplementation for prevention of ARI has demonstrated protective effects. Several cellular studies have investigated the effects of vitamin D metabolites on immune responses to respiratory viruses, but syntheses of these reports are lacking. Scope: In this article, we review the literature reporting results of  Viral acute respiratory infections (ARI) are a leading cause of morbidity and mortality worldwide, and, as such, are a major global health problem. They are also responsible for a huge economic burden, precipitating considerable absence from work and school, and large numbers of visits to clinicians.The viral pathogens most commonly associated with acute respiratory infections are picornaviruses (including the species rhinovirus (RV) A, RVB, RVC and the enteroviruses A–D), orthomyxoviruses , paramyxoviruses (including the parainfluenza viruses (PIV) 1–4, respiratory syncytial virus (RSV) and human metapneumovirus (hMPV)), coronaviruses, adenoviruses and human bocavirus (HBoV) ,3,4,5. TVitamin D is an immunomodulatory micronutrient ,12. As rin vitro studies have investigated the effects of vitamin D metabolites on host immune responses to respiratory viruses, but recent syntheses of this literature are lacking. In order to review these studies the PubMed database was searched using the terms “vitamin D” with the following respiratory viruses  to ensure a systematic review of the available literature. Inclusion criteria were studies which provided in vitro evidence as opposed to solely clinical studies, the availability of the full text, and for virus genera, such as enterovirus, which can infect multiple sites, the use of species or serotypes specifically associated with respiratory disease.A number of When a respiratory virus is inhaled it first binds to non-specific receptors on the respiratory epithelium, usually glycolipids or glycoproteins such as intercellular adhesion molecule (ICAM)-1. Membrane fusion or endocytosis follows, thus internalizing the virus and enabling subsequent replication, transcription and translation of new viruses which can then be released to infect new cells. However, once a cell has been infected, pathogen-associated molecular patterns (PAMPs) on the virus can be recognised by various intracellular innate pathogen recognition receptors (PRRs) such as the toll-like receptors (TLRs), retinoic-acid-inducible gene-I (RIG-I)-like receptors (RLRs) and nucleotide binding-oligomerisation domain (NOD)-like receptors (NLRs). Pulmonary epithelial cells have been shown to express all of the known human TLRs and RLRs which detect viruses, and ligands for these PRRs activate epithelial cells in order to initiate a rapid immune response against viral invasion . In addiThe intracellular TLRs 3, 7, 8 and 9 are mainly located on the endoplasmic reticulum (ER) membrane before UNC93B1-dependent (an ER multi-transmembrane-domain-containing protein) trafficking to the endolysosome following viral infection ,33. ThesUpon PAMP-PRR interaction and the activation of signalling transduction pathways, the type I IFNs (IFN-α and IFN-β) are some of the earliest cytokines to be produced. Their transcription and subsequent binding to the IFN receptor induces expression of a variety of interferon-stimulated genes (ISGs), with their products altering antiviral and immunomodulatory actions to limit and clear infection . FeatureInnate PRR signalling also results in the production of the chemokines CXCL8, CXCL9, CXCL10 and IL-15 which facilitate recruitment of neutrophils and NK cells, respectively ,49. Whil+ and CD8+ T lymphocytes. These T-lymphocytes are activated, proliferate, differentiate into effector T-cells and migrate via efferent lymph vessels into the circulation. Multiple chemokines, such as CCL3, CXCL9 and CXCL10, are expressed in the respiratory epithelium and result in changes in integrin affinity, allowing effector T-cells to bind to the endothelium and migrate into the infected tissue [+ effector T-cells and NK cells can then induce apoptosis of infected cells via the release of cytolytic granules or by direct interaction between surface Fas receptor and Fas ligand (FasL) [+ T-cells and naive B-cells in secondary lymphoid tissues results in their proliferation and antibody class-switching, with neutralizing virus-specific antibodies crucial for optimal viral clearance. Additionally, viral components expressed on infected cells allow antibodies to bind, thus initiating antibody-dependent cell-mediated cytotoxicity (ADCC), whereby CD16 on NK cells recognises the Fc portion of antibodies bound to the surface and kills the target cell [PRR signalling facilitates the maturation and trafficking of dendritic cells, with the release of the chemokines CCL2 and CCL20, and increased expression of CCR7 ,60. Thusd tissue ,62,63,64d (FasL) . B-cellsget cell ,65,66,67get cell . The evoget cell ,69. Ther3. This 25(OH)D3 is the circulating form of vitamin D, with a half-life estimated to range from two weeks to two months [2D3 and ligation of nuclear vitamin D receptors (VDRs), allowing subsequent binding to vitamin D responsive elements (VDREs) in promoter regions of specific genes, resulting in the repression or induction of gene transcription [2D3-mediated non-genomic rapid responses via the activation of second messenger systems, such as phospholipase C, protein kinase C and phosphatidylinositol-3′-kinase (PI3K), thus initiating various intracellular effects, such as the opening of voltage-gated calcium channels and Ras/MAPK signal transduction [3 levels seen with autoimmune diseases, cancer, cardiovascular disease and respiratory infections [Vitamin D  is acquired from dietary sources, primarily oily fish, or from UV-mediated synthesis in the skin, before it is metabolised in the liver to form 25(OH)Do months ,71,72, acription  D, infection by respiratory viruses resulting in recognition by TLRs and cytokine production is able to increase levels of 1,25(OH)2D, hypothetically altering the immune response to better respond to these pathogens.Therefore, extra-renal 1,25(OH)2D activity. Cathelicidins are multifunctional antimicrobial peptides, and the sole form in humans is Human Cationic Antimicrobial Peptide of 18KDa (hCAP-18). From this precursor, the active form of LL-37 is cleaved by serine protease 3 in neutrophils [2D-ligated VDR plays in binding to a VDRE in the promoter of the cathelicidin gene to enhance hCAP-18 production [Vitamin D has diverse and extensive effects on the immune system, due to the expression of the VDR and the enzyme CYP27B1 by most immune cells ,81,106. trophils . While ttrophils ,108, andtrophils . It has trophils ,114,115,trophils ,54,56. Noduction ,118,119,2D to upregulate expression of β-defensin 2 [The proximal promoter region of DEFB4 gene also contains a VDRE, allowing 1,25(OH)fensin 2 . This isfensin 2 ,121.2D to induce monocyte autophagy has also been demonstrated. Autophagy acts as part of the immune system to remove damaged proteins and organelles, and is an important host defence mechanism against viral infections [In addition to antimicrobial peptide induction, vitamin D has been demonstrated to modulate the innate immune system in a variety of other ways. Monocyte differentiation into macrophages is induced , with enfections . Vitaminfections ,138,139.2D [2D has been demonstrated to induce up-regulation of TLR2 in keratinocytes, allowing a mechanism by which vitamin D may prevent infection of wounds [2D in myeloid and epithelial cells, via two distal VDREs in the NOD2 gene. Addition of MDP  to 1,25pression .Vitamin D also modulates the adaptive immune response, and acts as a key intermediary between innate and adaptive immunity due to its influence on antigen presentation.2D has been demonstrated to inhibit DC differentiation, maturation and antigen presentation, with an associated decrease in markers such as CD1a, MHC class II, and the co-stimulatory molecules CD40, CD80 and CD86 [Dendritic cells are the most potent antigen-presenting cells, and, as such, have a direct effect on lymphocyte activation and induction of the adaptive immune response. They reside in peripheral tissues in an immature state, sampling the environment and mediating antigen uptake, until a maturation signal induces migration to local lymph nodes and subsequent T-cell activation. Addition of 1,25(OH)and CD86 ,147,148,and CD86 . Alreadyand CD86 .2D, with a decrease in MHC class II, CD40, CD80 and CD86. IL-12 production is suppressed in both activated DCs and macrophages, due to the 1,25(OH)2D-mediated down-regulation of NF-κB activation [2D administration in LPS-stimulated monocytes and PBMCs [The antigen-presenting and T-cell stimulatory capacity of monocytes and macrophages is also impaired by 1,25(OH)tivation , while ind PBMCs ,152.2D on DCs has a major impact on T-cells. The decreased surface expression on DCs of co-stimulatory molecules and MHC class II results in a tolerogenic phenotype, with DC production of IL-12 (which is involved in driving Th1 differentiation) and IL-23 (which is involved in driving TH17 differentiation) inhibited by 1,25(OH)2D [2D) reducing IL-17 production in a mouse colitis model and impairing commitment to the Th17 lineage in mice with experimental autoimmune uveitis [The main function of DCs is to initiate T-cell responses, and thus the effect of 1,25(OH)25(OH)2D ,150. Eve25(OH)2D ,153. IL-25(OH)2D . Concomi25(OH)2D ,156. Thi25(OH)2D . Th17 ce uveitis ,158, alt uveitis , deficie uveitis . Therefo2D has also been demonstrated to have direct effects on T-cells, independent of DC activity. While the role of DCs in the induction of Tregs has been described, it has also been shown that 1,25(OH)2D in combination with dexamethasone can induce a Treg population in the absence of APCs [2D. Production of IL-2, IFN-γ, TNF-α, IL-17 and IL-21 are all inhibited [2D is also able to upregulate the Th2-specific transcription factors GATA-3 and c-maf, resulting in increased production of IL-4, IL-5 and IL-10 [2D is able to inhibit proliferation, plasma-cell differentiation, immunoglobulin secretion and memory B-cell generation, while inducing B-cell apoptosis [+ T-cells from MS patients secreting less IFN-γ and TNF-α and more IL-5 and TGF-β following 1,25(OH)2D treatment [1,25(OH) of APCs . The pronhibited ,162,163,nhibited . This dind IL-10 . B-cellsnd IL-10 . 1,25D, DCs are required to convert this precursor to the active 1,25(OH)2D to exert its immunomodulatory effects [However, the mechanisms behind any potential beneficial role of vitamin D are unclear, with conflicting cellular studies on the effects of vitamin D on Th2 cells ,171, witgillosis , thus cogillosis  would su effects . This inThe main immunomodulatory effects of vitamin D are summarised in in vitro studies have described the general effects of vitamin D metabolites on the function of immune cells and secretion of inflammatory molecules, as described above, experiments investigating the specific antiviral effects of vitamin D on each respiratory virus are limited. The available literature is summarised below, and presented in While a number of 2D and subsequently infected with RV-16. Incubation with vitamin D metabolites had no effect on viral replication, and, whilst RV-16 infection induced hBEC secretion of CCL5, IL-6 and IL-29, incubation with vitamin D metabolites did not alter this effect. Treatment with vitamin D did, however, enhance secretion of CXCL8 and CXCL10, both in the presence and absence of rhinovirus infection. These molecules are pro-inflammatory chemokines, responsible for the recruitment of immune cells such as neutrophils, macrophages and T-cells to the site of infection, and could thus act as an effector mechanism as to how vitamin D alters the antiviral response to rhinovirus infection.Rhinovirus is the most common aetiologic agent of the common cold, which in turn is the most frequent acute infection in the industrialised world, associated with two to three episodes per year in adults, and five to seven per year in children ,176,177.Fokl SNP) and vitamin D binding protein (the GC1s variant) associated with a genetic predisposition to RSV bronchiolitis [in vitro work investigating the specific effects of vitamin D on the immune response to RSV is limited.RSV is usually associated with illness in young children, with most infants having been infected by the age of three, causing a mild URI for the majority, but severe bronchiolitis in some ,180. It hiolitis ,185,186.2D, expression of the NF-κB inhibitor IκBα was upregulated [2D prior to RSV infection IκBα levels were increased due to augmented mRNA transcription and protein synthesis. An increase in IκBα results in NF-κB remaining inactive in the cytoplasm, preventing its translocation into the nucleus and subsequent binding to DNA promoter regions. Thus, vitamin D inhibits RSV induction of NF-κB-driven IFN-β and CXCL10 secretion, as also demonstrated in this study. Additionally, interferon stimulated genes (ISGs) without functional NF-κB sites in their promoters were also measured, with 1,25(OH)2D treatment resulting in decreased levels of human myxovirus resistance protein 1 (MxA) and ISG15. This was demonstrated to be a consequence of the suppression of NF-κB-driven IFN-β production. Similarly, pre-treatment with 1,25(OH)2D before inoculation with RSV resulted in decreased levels of STAT1 protein and inhibition of STAT-1 translocation to the nucleus, also as a consequence of suppression of NF-κB-driven IFN-β production. However, even though production of important components in the antiviral response to RSV infection  were reduced by treatment with 1,25(OH)2D, viral replication and viral load was not increased. Thus, via increasing IκBα levels, 1,25(OH)2D is able to dampen the inflammatory response to RSV infection whilst maintaining the antiviral state and without having adverse effects on viral load, suggesting a potential role in reducing immunopathology. A similar study using alveolar A549 cells infected with RSV has also demonstrated that 1,25(OH)2D is able to decrease levels of phosphorylated STAT-1 (pSTAT-1) and increase IκBα protein levels, with subsequent diminished expression of interferon regulatory factor 1 (IRF1), IRF7, IFN-β and CXCL8 mRNA [FokI VDR variant, which has been associated with severe RSV bronchiolitis. No differences were observed between the two variants in viral replication, or the vitamin D-induced increase in IκBα and repression of NF-κB-regulated genes. However, in M1 FokI VDR-expressing cells, vitamin D was not able to repress expression of the STAT-1 regulated genes IRF1 and IRF7, due to decreased regulation of STAT-1 phosphorylation. Therefore, together these studies indicate that vitamin D is able to dampen NF-κB and STAT-1 mediated inflammation to reduce RSV immunopathology, with an impairment in the regulation of STAT-1 phosphorylation resulting in more severe disease.In a study using primary human tracheobronchial epithelial (hTBE) cells treated with 1,25(OH)egulated . Cells iCL8 mRNA . This stIt has also been demonstrated that the antimicrobial peptides LL-37 and β-defensin 2 have anti-viral activity against RSV, blocking viral cellular entry, preventing virus-induced epithelial cell death, inhibiting production of new infectious particles and diminishing the spread of infection ,121. SinFinally, a number of studies have been carried out in calves, looking at the association between vitamin D and bovine RSV (BRSV). Naturally occurring BRSV infection in young ruminants has been demonstrated to mimic the pathogenesis and lesions observed in RSV infection of infants, thus providing a useful model for studying RSV infection. Conversely to the effects described above, whereby production of NF-κB-linked pro-inflammatory cytokines was inhibited, vitamin D treatment has been shown to elevate secretion of the pro-inflammatory cytokine IL-12p40 following BRSV infection . No effein vitro work investigating the specific effects of vitamin D on the immune response to influenza viruses is lacking.The influenza virus is associated with seasonal epidemics, severe pandemics, pneumonia and secondary bacterial infections. As such, a simple and cost-effective prevention strategy is desirable, with clinical trial results showing promising effects of vitamin D supplementation in the prevention of seasonal influenza A infection in Japanese schoolchildren . However2D on infection with influenza A H1N1 was assessed [In a study using alveolar A549 cells, the effect of pre- or post-treatment with 1,25(OH)assessed . It was assessed ,188, treassessed ), this sThe antimicrobial peptides LL-37 and human β-defensin 2 have also been shown to have anti-viral properties against influenza virus, reducing disease severity and viral replication, inhibiting infectivity, and demonstrating neutralising activity ,193. As Finally, the majority of work into vitamin D and influenza infection has been investigating the potential use of vitamin D as an adjuvant in influenza vaccines. However, vitamin D has not been shown to be able to modulate the humoral response to inactivated influenza virus in this setting ,197,198.in vitro studies have been carried out to determine effects of vitamin D on the immune response to these pathogens. This is the case for human metapneumovirus, parainfluenza, adenovirus, coronaviruses, enterovirus and human bocavirus.In the case of other common viral causes of ARI in humans, no in vivo [in vitro studies performed to date have not shown that vitamin D metabolites inhibit viral replication in epithelial cells. However, all the studies reviewed demonstrated effects of vitamin D metabolites on expression and secretion of pro-inflammatory cytokines and chemokines. Interestingly, the effects of 1,25(OH)2D on chemokine expression and secretion varied between pathogens; this finding complements those of clinical trials suggesting that vitamin D-inducible responses may be more effective against some pathogens than others [in vitro actions of vitamin D in viral respiratory infection , and to clarify the effects of vitamin D metabolites on respiratory virus-induced expression of cell surface markers mediating viral entry and bacterial adhesion to respiratory epithelial cells.This review has identified a modest number of laboratory studies investigating immunomodulatory effects of vitamin D metabolites in respiratory epithelial cells infected with respiratory viruses. Despite clinical evidence that vitamin D supplementation may reduce susceptibility to such infections in vivo , in vitrn others . More st


The erbB receptors, including the epidermal growth factor receptor (EGFR), erbB2 , erbB3 (or HER3), and erbB4 (or HER4), are often aberrantly activated in a wide variety of human cancers. They are excellent targets for selective anti-cancer therapies because of their transmembrane location and pro-oncogenic activity. While several therapeutic agents against erbB2 and/or EGFR have been used in the treatment of human cancers with efficacy, there has been relatively less emphasis on erbB3 as a molecular target. Elevated expression of erbB3 is frequently observed in various malignancies, where it promotes tumor progression via interactions with other receptor tyrosine kinases (RTKs) due to its lack of or weak intrinsic kinase activity. Studies on the underlying mechanisms implicate erbB3 as a major cause of treatment failure in cancer therapy, mainly through activation of the PI-3 K/Akt, MEK/MAPK, and Jak/Stat signaling pathways as well as Src kinase. It is believed that inhibition of erbB3 signaling may be required to overcome therapeutic resistance and effectively treat cancers. To date, no erbB3-targeted therapy has been approved for cancer treatment. Targeting of erbB3 receptor with a monoclonal antibody (Ab) is the only strategy currently under preclinical study and clinical evaluation. In this review, we focus on the role of erbB3-initiated signaling in the development of cancer drug resistance and discuss the latest advances in identifying therapeutic strategies inactivating erbB3 to overcome the resistance and enhance efficacy of cancer therapeutics. ErbB3 (HER3) belongs to the erbB (HER) receptor family of type I receptor tyrosine kinases (RTKs). The erbB receptor family consists of the epidermal growth factor receptor (EGFR), erbB2 (HER2/neu), erbB3 (HER3), and erbB4 (HER4). It is arguably one of the most important receptor families in the context of development and tumorigenesis ,2. Abnorhttp://www.clinicaltrials.gov), because it lacks appreciable kinase activity [in vitro and in vivo[ErbB3 is frequently co-expressed with other RTKs in cancer cells. It promotes tumor initiation and progression mainly through activation of the oncogenic PI-3 K/Akt signaling. The erbB3/PI-3 K/Akt pathway is a major cause of treatment failure in cancer therapy because of its role in therapeutic resistance . It alsoactivity ,11. Seved in vivo-25. ThisErbB3 receptors are not capable of forming homo-dimers, but can induce activation of multiple downstream signaling pathways, such as PI-3 K/Akt, MEK/MAPK, PLCγ/PKC, Jak/Stat, and Src kinase, via hetero-dimerization with another RTK ,26. StuderbB3 are frequently observed in various malignancies, such as cancers of breast, gastric, ovarian, prostate, and bladder, colorectal carcinoma, squamous cell carcinoma of the head and neck, and melanoma [erbB3 occurring in approximately 11% of colon and gastric cancers [erbB3 are upregulated. Most metastatic breast cancers show expression of either EGFR or erbB2, whereas upregulation of both is not typical [erbB2/neu-transgenic mice [Amplification and/or overexpression of melanoma ,33,34. A cancers . Similar cancers . In brea typical . In cont typical  and brea typical . We and nic mice ,40. ErbBnic mice ,16. In enic mice ,42.Elevated expression of erbB3 protein has been reported in 50-70% of human breast cancers -45, and Of the four erbB receptors, erbB3 is best suited to activate the PI-3 K/Akt signaling, because it has the most tyrosine residues on its C-terminal tail once being phosphorated, they are capable of binding to the p85 subunit of PI-3 K ,57. In fin vitro and in vivo[Activation of erbB family members has been linked to tamoxifen resistance of estrogen receptor (ER) positive (ER+) breast cancers ,62. Crosd in vivo. In prosd in vivo. It has d in vivo. Thus, ede novo or acquired resistance to cetuximab-based therapy has erbB2 amplification or high levels of circulating HRG, which induces activation of erbB3 signaling [MET[erbB3 has been shown to involve in resistance to RAF/MEK inhibitors in the treatment of melanoma and thyroid carcinomas [erbB3 expression through the transcription factor, FOXD3 [erbB3 transcription via decreased promoter occupancy by the transcriptional repressors C-terminal binding protein 1 and 2 (CtBP1/2) [The EGFR- and/or erbB2-targeted therapies approved by FDA can be divided into two groups: 1) the blocking Abs, such as cetuximab (Erbitux) and panitumumab (Vectibix) against EGFR and trastuzumab (Herceptin), Pertuzumab (Perjeta), and T-DM1 (Kadcyla) against erbB2; 2) the small molecule tyrosine kinase inhibitors (TKIs), such as gefitinib (Iressa) and erlotinib (Tarceva) targeting EGFR and lapatinib (Tykerb/Tyverb) dual-targeting both EGFR and erbB2. All of these have been successfully used to treat a variety of human cancers. The majority of cancers develop resistance to these therapeutic Abs and/or inhibitors within one year. Numerous studies indicate that activation of erbB3 signaling is one of the major mechanisms of this resistance ,69. For ignaling . The erbling [MET. A recenling [MET. In addircinomas ,72. It ar, FOXD3 , whereasCtBP1/2)  or MAPK CtBP1/2) ,72. In lActivation of the survival signaling - PI-3 K/Akt pathway by erbB3  also gives rise to chemoresistance in cancer treatment. Docetaxel-based chemotherapy has been established as the standard of care for mCRPC. However, only half of the patients benefit from docetaxel. Of these, the majority will become resistance and eventually die of mCRPC ,73. MecherbB2 aberrant breast cancer. We have published a series of articles [In the last several years, our laboratory has focused on studying the unique biology of erbB3 receptor in the development of articles ,32,75,76articles . The molarticles -79. Botharticles -82. Nonearticles . Thus, narticles -85. Nonein vivo and show promise as novel cancer therapeutics [http://www.clinicaltrials.gov). Recent studies have identified bispecific Abs dual-targeting of EGFR/erbB3 [in vitro and in vivo[It is clear that there is overwhelming evidence to support the importance of erbB3 signaling in cancer progression, particularly in therapeutic resistance followed by tumor recurrence . Concomiapeutics ,88. Bothapeutics . This stapeutics ,11, suggapeutics ,23 and cFR/erbB3  or erbB2FR/erbB3 , that exFR/erbB3 . In addid in vivo.in vitro, their combinatorial in vivo antitumor activity could be attributed to induction of both growth inhibition and apoptosis [in vitro and in vivo[MM-121 is a fully humanized anti-erbB3 monoclonal IgG2 Ab. It inhibits ligand-induced dimerization of erbB3 and erbB2 and subsequently inactivates the downstream signaling. MM-121 has been shown to exert antitumor activity in preclinical models of various human cancers ,23. At ppoptosis . Our datpoptosis . The safpoptosis . AMG-888poptosis . It alsopoptosis . Recentld in vivo,94. Becad in vivo,94, we serbB2/erbB3. Further characterization revealed that entinostat upregulated three erbB2/erbB3-targeting miRNAs  which acted in concert to inhibit erbB2/erbB3 translation [in vitro and in vivo.Most erbB3-targeted therapies under development aim to inhibit erbB3 signaling by targeting the extracellular domain of the receptor. Novel approaches targeting of erbB3 have been proposed. One of these, EZN-3920  is a high-affinity, locked nucleic acid (LNA) antisense oligonucleotide. It specifically downregulates erbB3 and demonstrates significant antitumor activity in mouse xenograft models of breast and lung cancer cell lines . Clinicanslation . Thus, mnslation  via funcResearch on erbB receptors has long been focused on dysregulation of tyrosine kinase activity of EGFR and erbB2 in human cancers. Recently, the role of erbB3 as an obligate partner and in primary and acquired resistance to cancer therapeutics has attracted considerable attention. Increased awareness of erbB3 function in cancer progression, particularly tumor recurrence following drug resistance has several implications for future directions of investigation. ErbB3 may be considered as a valuable biomarker to predict the efficacy of EGFR- and/or erbB2-targeted therapy in the treatment of NSCLC and erbB2+ breast cancer, respectively. Therapeutic targeting of erbB3 has been shown to be an effective way to conquer drug resistance and significantly enhance the antitumor activity of hormonal therapy, targeted therapy, chemotherapy, and radiotherapy. Although numerous studies have dramatically improved our understanding of the biologic features of erbB3 receptor in cancer biology, and advances have been achieved in developing Abs against erbB3 with therapeutic potential, a number of critical questions still exist. First, activation of erbB3 signaling not only confers drug resistance in cancer treatment, but also promotes tumor metastasis -103. TheFDA: Food and Drug Administration; RTK: Receptor tyrosine kinase; ER: Estrogen receptor; PR: Progesterone receptor; AR: Androgen receptor; EGFR: Epidermal growth factor receptor; HRG: Heregulin; IGF-I: Insulin-like growth factor-I; IGF-1R: IGF-I receptor; PTEN: Phosphatase and tensin homolog; PI-3 K: Phosphoinositide 3-kinase; MAPK: Mitogen-activated protein kinase; MEK: MAPK kinase; AWT: Androgen withdrawal therapy; Ab: Antibody; TKI: Tyrosine kinase inhibitor; CRPC: Castration-resistant prostate cancer; mCRPC: Metastatic CRPC; NSCLC: Non-small cell lung cancers; IHC: Immunohistochemistry; CtBP1/2: C-terminal binding protein 1 and 2.The authors declare that they have no competing interests.JM, HL, JH, and BL drafted and finalized the manuscript. All authors read and approved the final manuscript.