The ability of lymphocytes and macrophage-derived cytokines and chemokines to modulate the activation of stromal cells during immune responses is well-documented, but few studies have investigated whether liver myofibroblasts shape the phenotype and function of monocytes in liver disease. In the present study, Kupffer cells were demonstrated to be activated in the inflamed livers of patients with cirrhosis and be in close contact with liver myofibroblasts. The Kupffer cells from cirrhotic livers expressed significantly elevated levels of PD-L1 , TLR4, CD80, CD32 and CD64 relative to those from normal livers. Consistent with this finding, the expression of these surface molecules was significantly upregulated in monocytes following exposure to liver myofibroblasts originating from inflamed livers. Accordingly, the liver myofibroblast-exposed monocytes exhibited a significant increase in dextran endocytosis. These data reveal that bidirectional interactions between liver myofibroblasts and Kupffer cells may function as an ‘amplification loop’ to enhance inflammation further in the liver. Liver myofibroblasts are central in the pathogenesis of liver diseases and should be considered as targets for the rational design of effective immune-based anti-inflammation therapies. Furthermore, it was also demonstrated that skin fibroblasts were as effective as liver myofibroblasts at inducing monocyte activation, suggesting that fibroblasts, which are numerous in the body, may represent an underrated cell population that is actively involved in immunomodulatory functions. Kupffer cells, the resident hepatic macrophages, are key in hepatic fibrogenesis and are targets of proinflammatory mediators –3. KupffLiver myofibroblasts (LMFs), which are principally derived from activated HSCs , are ablThe ability of lymphocytes and macrophages to modulate the activation of stromal cells during immune responses is well documented ,14, althThe phenotypes of Kupffer cells in cirrhotic livers were observed in the present study and the association between LMFs and Kupffer cells was investigated. Kupffer cells were shown to be activated, with a concomitant increase in the expression of certain surface molecules in fibrotic livers, while LMFs regulated the phenotype and function of monocytes through specific soluble factors. These results suggest that myofibroblasts are directly involved in regulating the function of monocytes in the human liver and that bidirectional interactions are present between the monocytes and LMFs in the liver microenvironment.Human specimens were obtained from patients attending the Sun Yat-sen University affiliated hospitals following approval by the ethics committee and receiving informed patient consent. The cirrhotic liver tissues were obtained from patients undergoing transplantation for alcoholic liver disease, hepatitis B- or C-associated cirrhosis, drug-induced liver disease, Wilson’s disease or cryptogenic cirrhosis. The liver tissues from patients with hepatic hemangioma were used as the normal controls.μm sections which were subsequently stained with polyclonal mouse anti-human CD68  primary antibody and rabbit anti-human α-smooth muscle actin (α-SMA) and fibroblast activation protein (FAP) primary antibodies  followed by Alexa Fluor 488- or 568-conjugated goat anti-mouse IgG and Alexa Fluor 568- or 488-conjugated goat anti-rabbit IgG  secondary antibodies. The positive cells were detected by confocal microscopy.For the immunofluorescence analysis, the liver tissues were cut into 5-LMFs and normal skin fibroblasts were isolated as described previously . The LMFThe LMFs were cultured on collagen-coated coverslips  and were fixed with 1:1 acetone/methanol for 10 min, rinsed and prewetted with phosphate-buffered saline (PBS)/10% fetal calf serum (FCS)/0.1% sodium azide. Next, the cells were stained with antibodies against fibroblast surface protein (FSP), vimentin, FAP, desmin, α-SMA, fibronectin and immunoglobulin G  in Tris-buffered saline (pH 7.4) for 60 min. The cells were washed and incubated for 20 min in isotype-relevant goat anti-mouse fluorescein-isothiocyanate (Invitrogen) and the nuclei were counterstained with 4′,6′-diamidino-2-phenylindole hydrochloride . The images were viewed and assessed using a fluorescence microscope (LEICA DMI 4000B) and analyzed using the Leica Application software suite (version 4.0).μm mesh and separated by Ficoll centrifugation. The mononuclear cells were washed and resuspended in media supplemented with 1% heat-inactivated FCS for the flow cytometry analysis.Peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coats derived from the blood of healthy donors using Ficoll density gradients, as described previously . The mon5 cells per well) in the presence of either LMFs or skin fibroblasts (monocyte/LMF or skin fibroblast ratio: 5/1). At the indicated time intervals, the monocytes were harvested, counted and analyzed.The monocytes were cultured in DMEM (Sigma) with 10% FCS in 48-well flat-bottom microtiter plates , containing 2 mmol/l L-glutamine, 60 μg/ml benzylpenicillin and 100 μg/ml streptomycin . The Multiplex bead-based enzyme-linked immunosorbent assay analysis of the conditioned supernatants was performed using the Human 38-plex antibody bead kit, the Human 11-plex antibody bead kit and the Human 23-plex antibody bead kit  and was analyzed using a Luminex plate reader and Milliplex analyst software (Luminex 200 System).Supernatants were generated by seeding 5×10The peripheral blood monocytes, liver monocytes and LMFs were stained with fluorochrome-conjugated antibodies against PD-L1, PD1, CD14, CD16, CD23, CD32, CD64, CD80, CD86, TLR2, TLR4, CD166, CD90, CD29, CD73, CD13, CD44, CD105, CD31, CD45, CD34 and control antibodies , according to the manufacturer’s instructions. The cells were subsequently analyzed using multicolor flow cytometry.The results are expressed as the mean ± SEM. Normality was tested using the Shapiro-Wilk test and the normally distributed data were compared using paired t-tests for associated samples, analysis of variance or independent t-tests. Non-normally distributed data were compared using the Wilcoxon signed-ranks test for associated samples or the Mann-Whitney U test for independent samples. SPSS statistical software (version 13.0) was used for all the statistical analyses. Unless otherwise specified, all the data were analyzed using two-tailed tests and P<0.05 was considered to indicate statistically significant differences.+CD14+ cells  were demonstrated to be in close contact with the LMFs (highly expressing FAP), suggesting that the LMFs may regulate the function of Kupffer cells via certain signals . CollectA total of 7 primary LMF cell lines were established from patients with cirrhosis and the cell phenotypes were analyzed by immunofluorescence and cytofluorimetric analyses. As shown in To investigate the possible effect of LMFs on monocytes, the cells were cultured together. Monocytes, freshly isolated from the blood of unrelated healthy donors, were cocultured with various LMF cell lines for 3 or 6 days. The expression of surface molecules on the monocytes  was subsequently analyzed by flow cytometry. The results showed that the LMFs were potent at promoting the activation of the monocytes which exhibited phenotypic features similar to the monocytes isolated from the cirrhotic livers . It is nTo investigate the endocytotic ability of the monocytes following pretreatment with the LMFs further, FITC-dextran was supplied for 30 min in the coculture system. On days 3 and 6, the LMF-exposed monocytes demonstrated significant increases in dextran endocytosis . MoreoveTo obtain further insight into how the LMFs or skin fibroblasts were able to modulate the monocytic response, a method was designed to determine whether the enhancing effect was due to diffusible factors or required direct cell-to-cell interactions. After purified monocytes were cultured in conditioned medium from LMFs or skin fibroblasts, it was demonstrated that the supernatant from the fibroblasts also effectively induced the activation of monocytes, suggesting that certain soluble factors were secreted to modulate the monocytes (data not shown).Taken together, these results suggest that LMFs are critical in maintaining the activation of monocytes in the fibrotic liver environments of humans.in vitro cocultures of monocytes and LMFs/skin fibroblasts were established and their conditioned media were screened for levels of various cytokines, chemokines and growth factors using the Multiplex bead-based enzyme-linked immunosorbent assay. Notably, the levels of the majority of the cytokines, chemokines and growth factors in the cocultures were higher than those produced by the monocytes alone  in the liver ,23 whereThe liver consists of hepatic parenchyma and a large proportion of nonparenchymal cells (NPCs), including sinusoidal endothelial cells, Ito cells and dedicated hepatic macrophages (Kupffer cells) . Kupfferin vitro is likely to reflect their function in vivo(LMFs originate principally from activated HSCs. However, in fibrotic disease, subpopulations arise from other sources, such as bone-marrow precursors –29. Sincn in vivo. Differen in vivo. Consistn in vivo.Monocytes are versatile, plastic cells that respond to environmental signals through diverse functional programs ,33. OtheThe skin fibroblasts were as effective as the LMFs at inducing the activation of monocytes, suggesting that fibroblasts, which are numerous in the body, may represent an underrated cell population that is actively involved in immunomodulatory functions. However, the mechanisms involved in the activation of monocytes may differ between the LMFs and skin fibroblasts for a number of the distinct soluble factors in the coculture systems.The observation that LMFs modulate the phenotype and function of Kupffer cells provides a mechanism whereby LMFs may determine the immune status within the inflamed liver. There is a fine-tuned collaborative interaction between immune cells and LMFs in liver microenvironments. Indeed, bidirectional interactions between LMFs and Kupffer cells may function as an ‘amplification loop’ to enhance inflammation further in the liver, thereby extending the role of the LMFs in liver disease from fibrogenesis to an active role in regulating inflammation. Although these regulatory loops must be studied in more detail, the present study provides novel insights into the mechanisms underlying hepatic inflammation and particularly the role of LMFs as proinflammatory elements. Together with previous studies indicating that HSCs act as antigen-presenting cells , the fin


Setaria italica) have, until now, been poorly understood.MicroRNAs (miRNAs) are a class of short non-coding, endogenous RNAs that play key roles in many biological processes in both animals and plants. Although many miRNAs have been identified in a large number of organisms, the miRNAs in foxtail millet  had functional annotations in InterPro and GO analyses, indicating the targets of the miRNAs were involved in a wide range of regulatory functions and some specific biological processes. A total of 69 pairs of syntenic miRNA precursors that were conserved between foxtail millet and sorghum were found. Additionally, stem-loop RT-PCR was conducted to confirm the tissue-specific expression of some miRNAs in the four tissues identified by deep-sequencing.We predicted, for the first time, 215 miRNAs and 447 miRNA targets in foxtail millet at a genome-wide level. The precursors, expression levels, miRNA* sequences, target functions, conservation, and evolution of miRNAs we identified were investigated. Some of the novel foxtail millet miRNAs and miRNA targets were validated experimentally. MicroRNAs (miRNAs) are 20 ~ 22 nucleotide (nt) non-coding small RNAs (smRNAs). Most primary miRNAs (pri-miRNAs) are transcribed from miRNA genes by RNA polymerase II (RNApol II), while others are transcribed by RNA polymerase III (RNApol III) . In planDrosophila melanogaster, Caenorhabditis elegans, and some mammals [Arabidopsis thaliana and Oryza sativa[Mirtrons are a new, recently discovered, type of miRNAs that originate from spliced introns of gene transcripts. Mirtrons reside within the intronic regions of genes and are processed through a Drosha-independent pathway, making them quite distinct from the other miRNAs. The mirtron pathway was first identified in  mammals -12 and hza sativa,14. Receza sativa,16.In plant, miRNAs are known to play crucial roles in many developmental events  and reguA. thaliana, 401 in Populus trichocarpa, and 164 in Nicotiana tabacum have been discovered, and, in monocotyledonous species, 713 mature miRNAs in O. sativa, 16 in Saccharum offcinarum, 241 in Sorghum bicholor, 42 in Triticum aestivum and 321 in Zea mays have been reported . Although foxtail millet is an important cereal crop, only a few miRNAs in foxtail millet have been reported. Bennetzen et al. [With the advent of second generation sequencing technology, the rate of miRNA discovery has increased dramatically -25. In dn et al.  reportedFoxtail millet is a diploid C4 panicoid crop species . It is aArabidopsis[Arabidopsis.To identify miRNAs in foxtail millet, we sequenced smRNAs for shoots (14-day-old) by high throughput Illumina sequencing technology . Nearly abidopsis and riceabidopsis, respectA. thaliana[The overall size distribution of the unique reads from four sequencing efforts were very similar, with the 24 nt smRNAs being the most abundant, followed by the 23 nt and 21 nt smRNAs  in the smRNA datasets that have never been reported in other species. Because some of these miRNAs were derived from multiple precursors, these 172 nov-sit-miRNAs corresponded to 212 pre-miRNAs, 68% of which were located in intergenic regions. Only a few of the 212 pre-RNAs were located in introns or in the UTRs of coding genes, which is in agreement with the reported statistical characteristics of plant pre-miRNAs [Among the 172 nov-sit-miRNAs, nine were found in all four libraries, nine were in three libraries, 25 were in two and 129 were in one library. These potentially nov-sit-miRNAs had relatively lower expression levels than the 43 known sit-miRNAs. For example, in leaf, the total expression levels of the 172 nov-sit-miRNAs 2267 RPM) were less than 1% of the total expression levels of the 43 sit-miRNAs (38438 RPM) and, in flower, the total expression levels of all the nov-sit-miRNAs were only 659 RPM. These results were consistent with a study in rice that found that most of the non-conserved miRNAs exhibited tissue-specific expression patterns and had relatively low expression levels compared with known miRNAs [67 RPM weThe conservation of miRNA sequences across species could be regarded as powerful evidence to annotate miRNA . We idenTo predict mirtrons, we analyzed the secondary structure features of the introns of the foxtail millet genes that were annotated in the Phytozome database (see the Methods section for details). Introns can be folded directly to acquire stem–loop structures similar to the pre-miRNAs. Using RNAfold, we found many introns that had reasonable second structures; that is, a single stem-loop with less than 3 nt overhangs at both ends, and a minimum energy of -25 kcal/mol or less. After mapping the smRNAs in our datasets onto the selected introns using the BLAST algorithm (see the Methods section for detail), we found only a few perfectly matched smRNAs; therefore, only two mirtron precursor candidates could be identified (PAC: 19709968 and PAC: 19675165)  Table .About 79% (351) of the predicted target genes had functional annotations in InterPro, the integrated resource for protein families, domains, and functional sites  ,47. The To analyze whether or not miRNAs in different tissues have different functions, GO enrichment analyses were conducted for the predicted miRNA targets in each of the four tissues. We found that the functions of the targets were similar in all four tissues; the targets were all enriched in biological regulation, biosynthetic process, macromolecule metabolic process and cellular process. In addition, some of the predicted targets had annotations in the cellular component category, including cell, and cell part in leaf and flower. In the root dataset, the target genes were annotated with significantly fewer GO terms than the target genes in the other three tissues. Details of the GO annotations for the target genes in the four tissues are available in Additional file To confirm the accuracy and reliability of the smRNA-seq results, we randomly chose ten of the miRNAs (five known miRNAs and five novel miRNAs) for stem-loop RT-PCR validation Figure B. The reArabidopsis[Previous reports in abidopsis and in rabidopsis have shoabidopsis.To date, miRNAs expression and function has been well studied in the major food and feed crops, such as sorghum, maize and rice ,50-53. FBased on the total number of genes in foxtail millet and in sorghum, we found 919 syntenic blocks containing 28,510 pairs of syntenic genes between the two species. In total, 20,090 foxtail millet and 19,877 sorghum genes, and 65 foxtail millet and 57 sorghum pre-miRNAs, were found within the syntenic regions. Nearly all the pre-miRNAs were located in only 47 (5%) of the 919 syntenic blocks, but within these 47 blocks there were 12,757 syntenic gene pairs corresponding to 45% of the total number of syntenic genes in the two species. We identified 69 pairs of syntenic pre-miRNAs, and all the foxtail millet and sorghum chromosomes had blocks that contained at least one of the conserved syntenic pre-miRNAs . We found that the expression of the sit-miR172 family was higher in the adult leaf and flower datasets and lower in the young shoot dataset. In maize, the miR172 that is responsible for the transition from juvenile to adult was reported to have the same expression pattern . In addiogenesis . SimilarArabidopsis, miR160 and miR167 were found to target auxin response factor [We found that, except for a few of the sit-miRNAs that had relatively high expression levels, most of the sit-miRNAs, especially the nov-sit-miRNAs, had low expression levels in the four studied tissues. It has been suggested that the normal physical functions of an organism require various miRNAs working together for precise regulation and control rather than individual miRNAs working separately . If thise factor ,70,71. Ie factor . Most ofe factor ,72,73.A. thaliana, the GRF gene family (AtGRF) of proteins that are involved in cell expansion in leaf and cotyledon tissues [Arabidopsis and that its predicted target gene contained the same characteristic regions  and WRC  domains) as the AtGRF proteins. Compared with other novel miRNAs, the nov-sit-miR64 had a relatively high expression in leaf (88 RPM), suggesting that nov-sit-miR64 may play an important role in leaf; however, nov-sit-miR64 expression was highest in root (1518 RPM) compared with in the other three tissues (less than 90 RPM). These findings suggest that nov-sit-miR64 (or miR396) not only plays an important role in leaf growth, but may also be involved in the development of root. The expression pattern of nov-sit-miR64 was validated by experiment  and Napier grass (Pennisetum purpureum) [Foxtail millet is considered to be a diploid, tractable model for polyploid biofuel crops like switchgrass (rpureum) . The synrpureum) -78. In trpureum) , and lacThe expression pattern of some sit-miRNA families also showed similar trends as the corresponding conserved miRNAs in different plants. For example, the expression levels of sit-miR156, sit-miR164, sit-miR166 and sit-miR167 were comparatively very high, and sit-miR160, sit-miR319, sit-miR390 and sit-miR394 were comparatively very low, in agreement with similar findings in other plants ,22,80. BIn this work, we examined the expression profiles of ten miRNAs in four tissues, three of which appeared to not be very consistent with the smRNA-seq data, suggesting that preprocessing and normalizing the data to help in identifying differentially expressed genes is a process that continues to be developed.By smRNA sequencing, we identified 43 known miRNAs and 172 novel miRNAs in foxtail millet and studied their expression profiles in four different tissues. Potential targets were predicted with strict criteria as described, and four targets were validated by 5′RACE. The functional annotation provided a deeper understanding of the transcription and regulation of the target genes, confirmed many known regulatory mechanisms, and provided a window into many more potentially novel pathways. Furthermore, a comparative genomic analysis with sorghum contributed to understanding the evolutionary dynamics of miRNA family expansions and will serve as the basis for future scomparative functional genomic studies using syntenic analysis. The identification and characterization of miRNAs from foxtail millet will aid in further research on foxtail millet and other species in the Poaceae family of grasses.Foxtail millet inbred line Yugu1 ,27 was uThe foxtail millet genome sequences were downloaded from the Phytozome database . To find foxtail millet miRNAs, a previously reported workflow was used  with a sThen, for each sequenced RNA sample, short reads were first mapped to all the candidate precursors. The location of the reads was checked to identify and eliminate the reads that mapped on the loop region of the corresponding precursor. Finally, the most abundant reads that were 20 ~ 22 nt in length with no less than 5 reads were regarded as potential miRNAs. A split-screen view of read alignments from a shoot (14-day-old) sample displaying regions of ten miRNA precursors is shown in Additional file To identify known and conserved miRNAs in foxtail millet, BLASTN was used to match the candidate miRNAs to the mature miRNAs from all 14 of the previously investigated plant species  in miRBaTo identify miRNA*, the first step of our pipeline is using BLASTN to align smRNAs from our sequencing libraries to miRNA sequences. We identified miRNA* candidates by allowing no more than four mismatches between the smRNA sequences and the miRNA sequences. Then, miRNA precursors were folded by RNAfold and the predicted miRNA* sequences that have 2 nt 3′ overhangs to the mature miRNAs were selected by an in-house developed Perl script, which can be obtained by request. Finally, if the sequence of predicted miRNA* can be found among the miRNA* candidates in the BLASTN output, we defined it as miRNA* in our study.To find mirtrons in the foxtail millet, we obtained all the intron sequences from the annotation  and assembly  files (data downloaded from Phytozome) files in the Phytozome database. Because the lengths of plant pre-miRNA are usually between 60 ~ 500 nt, we selected the introns within this length range for secondary structure prediction using RNAfold . Sequenchttp://wego.genomics.org.cn/cgi-bin/wego/index.pl) [To predict the target genes of foxtail millet miRNAs more precisely, the foxtail millet gene set (release 164) and annotation data were downloaded from Phytozome. There were nearly 392,553 entries in the transcript sequence file. We used a target prediction method with a modified scoring system as described previously . Basicalndex.pl)  was usedE. coli DH10B cells. Sequencing was carried out by Majorbio . The primers that were used in this study are listed in Additional file cDNA templates were prepared from the total RNA that was extracted from the 14-day-old shoots using a GeneRacer kit (Invitrogen) according to the manufacturer’s instructions. For each miRNA target gene, two gene-specific primers (GSP1 and NGSP1) were designed using Primer Premier 5.0 software . These tTotal RNA from foxtail millet shoot (14-day-old) was extracted as described in miRNA target validation by 5′RACE. PCR was performed as follows: 94°C for 2 min, 40 cycles of 94°C for 15 s and 60°C for 1 min. The PCR products were analyzed by 4% agarose gel and the specificity of the amplification was validated by sequencing the products. The primer design and the stem-loop RT-PCR procedure were performed as described previously ,93. All ΔΔCT method [Total RNA was isolated from roots, leaves, flowers and shoots (14-day-old) using TRIzol Reagent  and digested with RNase-free DNase I  to remove genomic DNA contamination. About 100 ng of DNA-free total RNA was hybridized with a miRNA-specific stem-loop RT primer. The hybridized miRNA molecules were then reverse transcribed into cDNA as described ,93. RealT method  was used-3) was used to identify homologous protein-coding and pre-miRNA genes between foxtail millet and sorghum. (The sorghum pre-miRNA sequences were downloaded from miRBase release 20) DAGchainer [The protein-coding gene sequences of foxtail millet (release 164) and sorghum (release 79) were downloaded from Phytozome. BLASTN  Total reads of 18 ~ 31nt small RNAs distribution from four different tissues. (B) Overlap among four sequenced small RNA libraries.Click here for fileDetailed information of known miRNAs in foxtail millet.Click here for fileDetailed information of novel miRNAs.Click here for fileSecondary structure of novel miRNA precursors.Click here for fileThe detail information about predicted mirtron candidates.Click here for filePotential targets of all miRNAs. Annotations were retrieved from the InterPro database.Click here for fileThe miRNAs  validated by stem-loop RT-PCR and sequencing.Click here for fileGO functional enrichment analysis for predicted target genes of miRNAs in four tissues respectively with comparisons to total foxtail millet genes.Click here for fileSyntenic sites harboring MIR genes in foxtail millet and sorghum.Click here for fileSplit-screen view of read alignments from shoot (14-day-old) sample displaying regions of ten miRNA precursors.Click here for filePrimers used in this study.Click here for file


Aedes aegypti, a highly domesticated mosquito species that is notoriously difficult to control. When transinfected into Ae. aegypti, the intracellular bacterium Wolbachia has recently been shown to inhibit replication of DENVs, CHIKV, malaria parasites and filarial nematodes, providing a potentially powerful biocontrol strategy for human pathogens. Because the extent of pathogen reduction can be influenced by the strain of bacterium, we examined whether the wMel strain of Wolbachia influenced CHIKV and YFV infection in Ae. aegypti. Following exposure to viremic blood meals, CHIKV infection and dissemination rates were significantly reduced in mosquitoes with the wMel strain of Wolbachia compared to Wolbachia-uninfected controls. However, similar rates of infection and dissemination were observed in wMel infected and non-infected Ae. aegypti when intrathoracic inoculation was used to deliver virus. YFV infection, dissemination and replication were similar in wMel-infected and control mosquitoes following intrathoracic inoculations. In contrast, mosquitoes with the wMelPop strain of Wolbachia showed at least a 104 times reduction in YFV RNA copies compared to controls. The extent of reduction in virus infection depended on Wolbachia strain, titer and strain of the virus, and mode of exposure. Although originally proposed for dengue biocontrol, our results indicate a Wolbachia-based strategy also holds considerable promise for YFV and CHIKV suppression.Incidence of disease due to dengue (DENV), chikungunya (CHIKV) and yellow fever (YFV) viruses is increasing in many parts of the world. The viruses are primarily transmitted by  Aedes aegypti, a mosquito that due to its close association with humans has historically been difficult to control. An innovative control strategy involving the release of mosquitoes infected with the intracellular bacterium Wolbachia is currently being developed. This approach is based on the recent discovery that Wolbachia reduces infection of mosquitoes with dengue virus, malaria parasites and filarial nematodes. In the current study, we demonstrated that Wolbachia also blocks infection of chikungunya and yellow fever viruses in Ae. aegypti. The degree of virus inhibition depended on the strain of Wolbachia, the route of virus exposure, the virus strain and the titer of virus that the mosquitoes were exposed to. The implementation of Wolbachia-based control strategies has the capacity to transform the way that mosquitotransmitted diseases are controlled in the future.Mosquito-transmitted viruses such as dengue, yellow fever and chikungunya, are responsible for significant morbidity and mortality throughout tropical and sub-tropical regions of the world. These viruses are primarily transmitted by  Mosquito-transmitted viruses cause significant human morbidity and mortality throughout the world and impose particularly heavy health and economic burdens on developing countries. Dengue, caused by infection with any of the four dengue virus (DENV) serotypes, is currently the leading arboviral disease, with millions of cases of classic dengue fever and tens of thousands of deaths annually due to hemorrhagic disease Aedes aegypti, a highly domesticated species that feeds almost exclusively on humans. Its geographic range has expanded with increased urbanization, resulting in increased arbovirus transmission s-methoprene), or adulticiding with indoor residual spraying or ultra-low-volume application. While these approaches can be successful, they are often labor-intensive, can be prohibitively expensive to implement and require a sustained commitment from all levels of government Current disease control measures focus on the suppression of mosquito vector populations to reduce virus transmission. The primary vector of DENVs, CHIKV and YFV is the mosquito Ae. aegypti mosquitoes with Wolbachia pipientis from other insect hosts Wolbachia is a maternally transmitted, endosymbiotic bacterium that manipulates host reproduction to enhance its own transmission Wolbachia can provide its host with nutritional benefits Ae. aegypti transinfected with the wMelPop-CLA strain of Wolbachia from Drosophila melanogasterWolbachia-free control mosquitoes wMelPop-CLA may also impose fitness costs on Ae. aegypti, such as reduced fecundity due to poor blood feeding success An emerging biocontrol approach to reducing transmission of arboviruses is provided by the transinfection of Wolbachia, wMel that is closely related to wMelPop-CLA and also occurs naturally in D. melanogaster was recently introduced into Ae. aegypti as an additional strain for the biocontrol of dengue Wolbachia in natural populations of mosquitoes. Unlike wMelPop-CLA infected mosquitoes, Ae. aegypti infected with wMel did not suffer any significant deleterious fitness costs when compared to uninfected controls wMelPop-infected Ae. aegypti, wMel-infected mosquitoes displayed dramatically reduced replication of DENV-2 Ae. aegypti infected with wMel were deployed in the field in north Queensland, Australia over the 2010–2011 summer Wolbachia was able to reach almost 100% fixation in wild mosquito populations only a few months following release, indicating that Wolbachia-infected mosquitoes present a very promising strategy for the control of dengue that is cost-effective and poses minimal environmental and social harm A second strain of Ae. aegypti infected with wMelPop-CLA showed reduced infection with CHIKV wMelPop-CLA infected mosquitoes could potentially be used for biocontrol in areas where human pathogens other than DENVs occur. However, not all strains of Wolbachia protect equally well, with those phylogenetically most closely related to wMel and wMelPop conferring the greatest degree of protection Drosophila, wMel confers protection against a range of viruses, for example Drosophila C virus, Flock House virus and Cricket paralysis virus wMel infection is able to limit the replication of human pathogens other than DENVs in mosquitoes, information that is critical to evaluating different Wolbachia strains for biocontrol.Although originally developed as a biocontrol tool for DENVs, wMel strain of Wolbachia to limit infection in Ae. aegypti with CHIKV and YFV. We also tested the ability of YFV to replicate in mosquitoes infected with wMelPop-CLA, in order to compare this strain to wMel. We found reduced levels of CHIKV but not YFV infection in wMel-infected Ae. aegypti, although the degree of virus inhibition depended on the mode of infection. By contrast, mosquitoes harboring the wMelPop-CLA strain of Wolbachia showed reduced infection rates with YFV with the extent of reduction virus strain and titer dependent.Here, we tested the ability of the Ae. aegypti were used in the experiments. The transinfection of Ae. aegypti with the wMelPop-CLA and wMel strains of Wolbachia and maintenance of infected lines has been previously described wMel-infected line, MGYP2, and its tetracycline-treated counterpart, MGYP2.tet, were exposed to both YFV and CHIKV. Both lines were in the F16 generation for the YFV experiments and the F24 generation for the CHIKV experiments. The original wMelPop-CLA infected line, PGYP1 52 and F53 generations, while the PGYP1.tet was used in the combined F50 and F52 generations. A Wolbachia-uninfected wild-type line of Ae. aegypti, designated Cairns3, which originated from Cairns, Australia, was used as a positive control for all experiments. The YFV-susceptible Rex-D white-eye Higgs strain of Ae. aegypti that originated from Rexville, Puerto Rico, was used as an additional positive control for the YFV experiments Six different lines of Ae. albopictusAe. albopictus) cells.The CHIKV strain was isolated from a patient visiting Melbourne, Australia in 2006 and contained the alanine to valine mutation in the membrane fusion glycoprotein E1 gene (E1-A226V) that has been linked to increased infectivity in mosquitoes, especially Ae. aegypti lines were exposed to YFV via intrathoracic inoculation and in an infectious blood meal. However, low feeding rates in all lines coupled with unexpectedly low infection rates in control lines that did actually feed, compromised our ability to draw any meaningful conclusions from the YFV oral feeding data. Thus it was excluded from the current paper. For intrathoracic inoculation, immobilized mosquitoes were inoculated with 0.5 µl and 0.22 µl of YFV or CHIKV suspension, respectively. The suspension consisted of stock virus diluted in growth media  which contained antibiotics and antimycotics and was supplemented with 10% fetal bovine serum (FBS) for YFV or 3% FBS for CHIKV. For oral feeding, mosquitoes were allowed to feed on a blood meal consisting of CHIKV diluted in commercially available defibrinated sheep blood  and 1% sucrose. The blood meal was housed within a membrane feeding apparatus that was fitted with pig intestine as the membrane 2 and engorged mosquitoes placed in 900 ml gauze-covered containers. All mosquitoes were maintained on 10% sucrose at 28°C, high relative humidity and 12L∶12D light cycle within an environmental growth cabinet.The CHIKV experiments were undertaken in the Biosafety Level 3 (BSL-3) insectary at Queensland Health Forensic and Scientific Services, Brisbane, Australia, and the experiments with YFV were undertaken in the BSL-3 insectary located at the University of Texas Medical Branch, Galveston, Texas, USA. The MGYP2, MGYP2.tet and Cairns3 lines were exposed to CHIKV using both intrathoracic inoculation and oral feeding. Intrathoracic inoculation was employed because it circumvents the midgut infection and escape barriers, and allows a standard amount of virus to be delivered. The six For the YFV experiments, mosquitoes were processed at either day 10 or 14 post-exposure. Mosquitoes were chilled and the heads removed from the bodies and placed separately in 0.5 ml of GM supplemented with 10% FBS, and antibiotics and antimycotics. Recovery of virus from the head demonstrated that the virus had infected head tissue and was potentially able to be transmitted.in vitro capillary tube system 2 and the legs and wings removed. The saliva was collected by inserting the proboscis of the mosquito into a capillary tube containing GM with 20% FBS. After 30 min, the contents of the capillary tube were expelled into 600 µl of GM with 3% FBS. The salivary glands were then dissected from the body in a drop of GM with 3% FBS. The dissected salivary glands were washed in a drop of 1% bleach For the CHIKV experiments, infection, dissemination and transmission were assessed using a modified Salivary glands were also excised for visualization of CHIKV infection via an immunofluorescence assay (IFA). The glands were dissected in a drop of PBS and washed in another two drops of PBS, before each gland was placed in separate wells of an 18 well microscope slide. Slides were air-dried, fixed in cold acetone for 30 min, before being stored at −80°C.Blood/virus mixtures were titrated as serial ten-fold dilutions in 96 well microtiter plates seeded with confluent monolayers of C6/36 cells. Plates were incubated at 28°C for 5 days or 7 days for YFV- or CHIKV- infected blood/virus mixtures, respectively, before being fixed in PBS/acetone and stored at −20°C. Infection with YFV was detected using an indirect immunofluorescence assay with the monoclonal antibody (Mab) 863 Heads, salivary glands, bodies and body remnants were homogenized and clarified by centrifugation. The CC-EIA was used to detect CHIKV in all mosquito tissues and saliva expectorates. To further reduce bacterial and fungal contamination, the mosquito tissues and saliva expectorates were filtered through a 0.2 µm filter prior to being inoculated onto the C6/36 cell monolayers.Ct) value was <40. The number of YFV RNA copies in heads and bodies was quantified using the TaqMan RT-PCR, with RNA standards produced for the relative quantification of YFV RNA copy numbers normalized to RNA levels of the Ae. aegypti house-keeping gene RpS17 Salivary glands and saliva expectorates were also analyzed for CHIKV RNA using real time TaqMan RT-PCR. YFV viral RNA was detected in the heads and bodies using a YFV-specific TaqMan RT-PCR. Viral RNA was extracted from 140 µl of each of the mosquito components and the saliva expectorates using the QIAmp Viral RNA kit following the manufacturer's protocol , with modifications described in Moreira and others used for the YFV extractions 3; Dako, Glostrup, Denmark). A slide containing wells inoculated with Ross River virus, Barmah Forest virus, Sindbis virus and CHIKV infected cells was included as a positive control. Slides were examined under an Olympus CX41 fluorescent microscope .Fifteen microliters of B10 Mab was added to each well. After incubation at 37°C for 30 min in a humidified container, slides were rinsed in PBS pH 7.4 before being washed for 10 min in PBS pH 7.4. Slides were air-dried before the addition to each well of 15 µl Dako Anti-Mouse FITC Conjugate  in 0.04% Evan's Blue in PBS pH 7.4. Slides were incubated and washed as described above, followed by application of two drops of Dako fluroescent mounting medium  and uninfected mosquitoes (the tetracycline-treated MGYP2.tet and wild-type Cairns3 lines) with CHIKV in two replicate oral feeding experiments. In both experiments, fewer MGYP2 mosquitoes displayed CHIKV infection compared to MGYP2.tet and Cairns3 lines (P<0.01). Similarly, in experiment 2, CHIKV was detected in significantly fewer MGYP2 (30%) bodies compared to MGYP2.tet (70%) and Cairns3 (67%) lines (P<0.01).We challenged s3 lines Table 1.P<0.01) in experiment 1, but only when using the TaqMan RT-PCR assay (P<0.01), but not Cairns3 mosquitoes.Next, we used both CC-EIA and a CHIKV-specific TaqMan RT-PCR assay to track dissemination of the virus to the salivary glands and saliva following oral feeding experiments. CHIKV was detected in significantly fewer MGYP2 (20%) salivary glands compared to those from MGYP2.tet (80%) and Cairns3 (66%) lines . Twelve days following oral exposure, 27% (3/11) and 67% (6/9) of the MGYP2 and Cairns3 lines, respectively, were positive in the IFA. There did not appear to be any consistency in the localization of virus within infected salivary glands from either Ae. aegypti line  Wolbachia strains on body infection rates, using intrathoracic inoculation with two strains of YFV isolated from Nigeria (BA-55) and Bolivia (Cinetrop 28). Four Wolbachia-uninfected mosquito lines were used as controls: Cairns3, Rex-D, MGYP2.tet and PGYP1.tet. Due to high mortality rates in some lines, mosquitoes exposed to 103.5 TCID50/ml of YFV BA-55 were tested at 10 days post virus exposure. Mosquitoes exposed to104.5 TCID50/ml of YFV BA-55 and 104.0 TCID50/ml of YFV Cinetrop 28 were tested at 14 days post exposure.We tested the effect of 3.5 TCID50/ml) and high (104.5 TCID50/ml) titers of YFV strain BA-55, MGYP2 mosquitoes had the same high (100%) rates of body infection as MGYP2.tet, Cairns3 and Rex-D mosquitoes (P<0.001). PGYP1 mosquitoes also displayed significantly lower rates of body infection compared to MGYP2 mosquitoes (P<0.001). At the higher virus titer a similar number (100%) of PGYP1 mosquitoes was infected with YFV as for the PGYP1.tet, Cairns3 and Rex-D lines. A similar pattern was observed for the Cinetrop 28 strain of YFV, with mosquito body infection rates ranging from 96–100% and no significant differences among the mosquito lines, although this strain was tested at only one titer (104.0 TCID50/ml).At both low , at both low and high virus titers (P<0.001). At high virus titer, a higher (78%) number of PGYP1 mosquitoes had virus in the heads and no significant differences in infection rates were found across the mosquito lines. High head dissemination rates (84–100%) were found across all mosquito lines challenged with the Cinetrop 28 strain of YFV, with no significant differences among them.Next, we assessed the impact of the two strains of s titers Table 2.Wolbachia on the number of YFV RNA copies in Ae. aegypti bodies following intrathoracic inoculation with the strains BA-55 and Cinetrop 28, at three different titers. Overall, infection with both Wolbachia strains resulted in a lower median number of virus copies per body in Ae. aegypti compared to control lines. However, PGYP1 mosquito bodies consistently displayed several log fewer copies than MGYP2 lines  in challenges with YFV strain BA-55 at a titer of 103.5 TCID50/ml. However, virus levels were high in both MGYP2 and the Wolbachia-uninfected lines  and significantly less than PGYP1.tet, Cairns3 and Rex-D mosquitoes   but not the MGYP2.tet or Cairns3 lines    , although the number of virus copies was still high  , although virus was present in the majority of PGYP1 heads  when mosquitoes were challenged with the Cinetrop 28 strain  . Median Wolbachia-infected mosquitoes into natural populations has been proposed as a way of reducing DENV transmission while minimizing social and environmental harm Wolbachia-infected mosquitoes may prevent the transmission of other significant arboviruses. Here, we evaluated whether mosquitoes infected with the wMel strain of Wolbachia show limited infection with CHIKV and YFV. To compare the two Wolbachia strains, we tested whether YFV was able to infect and replicate in wMelPop-CLA infected mosquitoes.Mosquito-transmitted viruses such as DENVs, CHIKV and YFV pose a significant health risk for almost half of the world's population. The release of wMel showed significantly reduced rates of CHIKV infection and dissemination to the salivary glands compared to controls, but only in the oral exposure experiments. CHIKV also showed limited dissemination in wMelPop-CLA-infected mosquitoes following oral exposure Wolbachia may be useful candidates for release in CHIKV control programs. By contrast, YFV was much less likely to infect and disseminate in Ae. aegypti infected with wMelPop-CLA compared to wMel strains. The virus was also less likely to replicate in wMelPop-CLA infected mosquitoes, with very high virus loads detected in wMel-infected Ae. aegypti. Our experiments suggest that wMelPop-CLA infected mosquitoes may be the best candidates for YFV biocontrol programs, but were unable to determine the extent of virus replication following oral exposure rather than intrathoracic inoculation. Because virus inhibition with some Wolbachia-virus combinations does not appear to be complete, it is essential that epidemiological models be utilized to establish the threshold of virus inhibition necessary to minimize and prevent transmission in the field.Mosquitoes infected with wMel strain occurs at high levels in Ae. aegypti ovaries and salivary glands but it is not present in as many body tissues as the wMelPop-CLA strain wMel does not accumulate to high levels in the fat bodies, brain and thoracic ganglia, which are involved in secondary replication of arboviruses prior to infection of the salivary glands wMel-infected mosquitoes to allow dissemination to the salivary glands and potentially be transmitted to humans. The presence of wMel in the mosquito did not completely prevent the replication of CHIKV in Ae. aegypti salivary glands, as determined by cell-culture, real-time RT-PCR and IFA analysis.The Wolbachia-infected Ae. aegypti show significant immune upregulation Wolbachia infection activates the Toll immune pathway in Ae. aegypti and the production of antimicrobial peptides which inhibit DENV-2 Wolbachia infection in Drosophila, the endosymbiont's native host, also blocks DENV-2 replication despite the absence of immune upregulation Wolbachia-mediated interference with virus replication, it may not be the whole explanation Wolbachia may compete with arboviruses for key cellular components in situ hybridization has revealed that wMelPop-CLA infection is widespread throughout the body of infected Ae. aegypti, and is prevalent in tissues and organs associated with viral replication, including the fat bodies, brain and ommatidia wMelPop-CLA. As DENV-2 and YFV are closely related flaviviruses, it is possible that a similar pattern of virus exclusion is occurring with YFV in wMelPop-CLA infected cells and tissues. In vitro studies have shown that reduced virus replication correlates with the density of Wolbachia in the cell Wolbachia-mediated interference with virus replication.Host immune pre-activation has been proposed as an explanation for interference with virus replication because Wolbachia limits arbovirus infection and replication in Ae. aegypti has the potential to fundamentally alter vector control strategies wMel-infected Ae. aegypti in two communities near Cairns, northern Australia, resulted in >90% frequency within the population six weeks after the first release Wolbachia-based control strategy was initially proposed to control DENVs, previous work and the current study have demonstrated that releases of Wolbachia-infected Ae. aegypti to control DENVs may have the added benefit of reducing CHIKV and YFV transmission. Alternatively, Wolbachia-infected Ae. aegypti could be released to specifically reduce transmission of these viruses, irrespective of whether DENVs were circulating or not in an area.The discovery that Wolbachia is dependent on the strain of Wolbachia, the strain of virus and the mode of exposure to the virus. Higher YFV intrathoracic inoculation titers allowed more breakthrough of virus in wMelPop-CLA infected mosquitoes. Infection rates, accumulation and dissemination to the head also differed between BA-55 and Cinetrop 28 in the presence of wMelPop-CLA, suggesting that different virus strains may have different replication dynamics in Wolbachia-infected mosquitoes. The CHIKV experiments showed that the TaqMan RT-PCR was generally more sensitive than the CC-EIA in detecting the presence of virus. This was not unexpected, given that the TaqMan RT-PCR does not differentiate between RNA derived from live, packaged infectious virus particles, from that of dead or defective, non-infectious virus. Thus, our experiments illustrate the importance of carefully selecting virus titer, virus strain, mode of delivery and method of virus detection when assessing the potential utility of different Wolbachia strains for biocontrol.We have demonstrated that the level of virus blockage induced by Wolbachia-based control strategies, it will be essential to assess the virus blocking ability of different Wolbachia strains against different virus strains, and if necessary, identify additional Wolbachia strains that could be deployed in the field. Commensurate with this will be the need for ongoing monitoring of Wolbachia-infected Ae. aegypti populations to confirm that the virus-limiting phenotype is maintained in successive generations post release.As part of the implementation of


Inhaled corticosteroids (ICS) reduce exacerbation rates and improve health status but can increase the risk of pneumonia in COPD. The GLUCOLD study, investigating patients with mild-to-moderate COPD, has shown that long-term (2.5-year) ICS therapy induces anti-inflammatory effects. The literature suggests that cigarette smoking causes ICS insensitivity. The aim of this study is to compare anti-inflammatory effects of ICS in persistent smokers and persistent ex-smokers in a post-hoc analysis of the GLUCOLD study.Persistent smokers (n = 41) and persistent ex-smokers (n = 31) from the GLUCOLD cohort were investigated. Effects of ICS treatment compared with placebo were estimated by analysing changes in lung function, hyperresponsiveness, and inflammatory cells in sputum and bronchial biopsies during short-term (0–6 months) and long-term (6–30 months) treatment using multiple regression analyses.+, CD4+, and CD8+ cells were reduced by short-term ICS treatment in smokers only. In addition, sputum neutrophils and lymphocytes, and bronchial CD8+ cells were reduced after long-term treatment in ex-smokers only. No significant interactions existed between smoking and ICS treatment.Bronchial mast cells were reduced by short-term and long-term ICS treatment in both smokers and ex-smokers. In contrast, CD3Even in the presence of smoking, long-term ICS treatment may lead to anti-inflammatory effects in the lung. Some anti-inflammatory ICS effects are comparable in smokers and ex-smokers with COPD, other effects are cell-specific. The clinical relevance of these findings, however, are uncertain. Chronic obstructive pulmonary disease (COPD) is characterized by chronic, partially reversible airflow limitation 1 decline and improvement in airway hyperresponsiveness and health status + lymphocytes, mast cells and macrophages Inhaled corticosteroids (ICS) have anti-inflammatory effects in asthma. Clinical effects of ICS in COPD have been investigated widely, providing positive effects on exacerbation rates and health status especially in advanced stages In vitro studies have shown that oxidative and nitrative stress, as can be caused by cigarette smoking, inactivates histone deacetylase-2, which is involved in the suppression of activation of inflammatory genes There are several indications that cigarette smoking may induce resistance to the anti-inflammatory effects of ICS. Smoking asthmatics are less sensitive to corticosteroids than non-smoking asthmatics The GLUCOLD study provided evidence for both clinical and anti-inflammatory effects of long-term ICS treatment in COPD. So far, no studies have linked long-term clinical and inflammatory effects of ICS treatment with active smoking in COPD. The aim of the current study is to assess anti-inflammatory effects of ICS in persistent smokers and persistent ex-smokers with COPD by a post-hoc analysis of the GLUCOLD study.The study was approved by the medical etics committee of Leiden University Medical Center (LUMC) and by the medical etics committee of University Medical Center Groningen (UMCG). All patients signed written informed consent. The study is registered at ClinicalTrials.gov with identifier NCT00158847.All persistent smokers and persistent ex-smokers from the GLUCOLD cohort were included Patients were randomly assigned into four groups as described before 1), and the provocative concentration of metacholine causing a 20% fall in FEV1 (PC20) were measured. Sputum samples were obtained by the full sample method 1 was assessed by standardized procedures as well as the two-minute tidal breathing method to measure PC20 methacholine At baseline and after 6- and 30-month treatment, inflammatory cells in induced sputum and bronchial biopsies, postbronchodilator forced expiratory volume in one second  absolute changes after short-term ICS treatment (baseline to 6 months) and after long-term ICS treatment (6 to 30 months). For the analyses from baseline to 6 months, the FP 6-month, FP 30-month, and FP/S 30-month groups were combined. For the analyses from 6 to 30 months, the placebo group was combined with the FP 6-month group, and the FP 30-month group was combined with the FP/S 30-month group. The effects of ICS treatment in persistent smokers and persistent ex-smokers and the interaction of ICS with smoking were investigated in the total population by multiple regression models. All models were adjusted for gender and age. The analyses were performed with SPSS version 18.0.3 and a p-value <0.05 was considered statistically significant.The absolute changes between the measurements at baseline and 6 months, and between the measurements at 6 months and 30 months, were calculated for FEVOf the 101 patients who adhered to therapy (>70% medication use) 20 significantly in ex-smokers  were generally greater in former smokers So far, studies investigating the effect of smoking on corticosteroid responsiveness have been performed in asthmatics predominantly. Smoking asthmatics were shown to be less sensitive to corticosteroids than non-smoking asthmatics 20, FEV1) and inflammatory parameters, in the same direction as the main results We believe that the fact that we analysed patient groups containing exclusively persistent smokers or persistent ex-smokers, in addition to the long-term intervention and the combined clinical and inflammatory outcomes represent the strengths of the current study. There are some limitations as well. We did not find significant interactions of ICS treatment response and smoking. This can be a real observation, yet it may also be due to low power, caused by the reduction in number of participants since we excluded switchers in smoking status. This resulted in small numbers in some subgroups, e.g. only 6 ex-smokers using ICS for six months, and thus this asks for caution of providing firm conclusions. For instance, there were differential effects with regard to short- and long-term treatment effects on lymphocytes, which may particularly result from analysing small numbers of patients. However, the direction of effects of other cell types was comparable with both short- and long-term follow-up providing robust findings. Additionally, we have to take into account that combining treatment groups in the 6–30 months period might have affected the results, for example by the effect of steroid withdrawal in the control group. However, when the individuals who stopped FP after 6 months were excluded, the analyses showed the same direction of effects and still reached significance despite lower study power . We realHow can we explain that mast cell numbers were reduced by short- and long-term ICS treatment in smokers and ex-smokers in stratified analyses, whereas other cells were affected either in smokers or ex-smokers, or not affected at all, as for instance macrophages? One explanation may be that this reflects an epigenetic phenomenon + cells specifically were reduced in ex-smokers after long-term ICS treatment. An in vitro study by Kaur et al. on lymphocytes obtained by bronchoalveolar lavage in COPD patients showed that dexamethasone reduced IL-2 and INFgamma production after PHA/PMA stimulation + cells, which are important key players in COPD, are sensitive to e.g. histone modification induced by smoke exposure, whereas mast cells are not.Gizycki et al. found that mast cells were reduced with 3-month ICS treatment in COPD patients who had high packyears smoking and smoked on average 27 cigarettes per day We suggest that the clinical implication of our findings may be that, even in the presence of smoking, long-term ICS treatment may lead to anti-inflammatory effects in the lung. However, especially in COPD patients with frequent exacerbations, the balance between positive clinical and anti-inflammatory effects and side effects ; significant data (p<0.05) are presented in bold; FEV1, post, L = FEV1 after salbutamol expressed in liters; PC20 = provocative concentration of metacholine causing a fall in FEV1 of >20%; ‡ Cell counts/10−7 per m2 of subepithelium; § Cell counts×104 per mL. The absolute changes in bronchial cell counts and sputum cell counts were normalized by Ln-transformation.(DOC)Click here for additional data file.


For the first time, it was observed that downregulated Snail expression is correlated with a significant inhibition of the expression and activity of RhoA GTPase, as well as MMP-2. The present data provide evidence that Snail promotes tumor cell motility and angiogenesis which is mainly mediated through the regulation of RhoA activity. In conclusion, the present findings demonstrate a key regulatory role for Snail in breast tumor growth and progression.Several lines of evidence support an important role for Snail, a transcriptional factor, in breast cancer. Overexpression of Snail has been associated with breast cancer metastasis, although the specific role of Snail in the process remains unclear. To address this issue, the expression levels of Snail, RhoA and fibronectin, as well as MMP-2, were reduced in the breast tumor cell lines MDA-MB-231 and MDA-MB-435S, and their biological responses were studied  Breast cancer is the leading cause of cancer-related mortality in females worldwide . Primaryin vivo  were maintained in Dulbecco’s minimum essential medium (DMEM) supplemented with 10% FCS and 1% antibiotic/antimycotic solution. Polyclonal antibodies against Snail, E-cadherin, MMP-2 and β-actin, and monoclonal antibodies against fibronectin, RhoA, Rac1, Cdc42 and PECAM-1 were obtained from Santa Cruz Biotechnology, Inc. .Conventional paraffin-embedded tissue sections obtained from breast tumors and normal breast tissue were obtained from surgical specimens resected at the Tongji Hospital of the Huazhong Science and Technology University . The avidin-biotin complex immunoperoxidase method was used to study the levels of Snail and RhoA expression by immunostaining. The frequency of each protein was scored as the percentage of positive cells as follows: negative, <5%; weak, 5 to 25%; moderate, 25 to 50%; and strong, >50%.μg/ml G418 for 12 days. Clones were picked and expanded for an additional two months. Experiments with the transiently transfected cells were performed 72 h after the transfections.cDNA encoding the open reading frame of Snail was amplified and cloned into the pIRES2-EGFP vector  in the inverted direction to produce an antisense-Snail cDNA construct (AsSn). Transient transfection for GFP alone , GFP-fused forms of wild-type RhoA, dominant-negative (DN) N19-RhoA and activated (Act) V14-RhoA  were also transfected into MDA-MB-231 and MDA-MB-435S cells and were then selected using 800 Total RNA was extracted according to the TRIzol instructions (Invitrogen), then combined with an RNase-free DNase kit  and reverse transcribed with random primers. The resulting cDNA was used for PCR using SYBR-Green master PCR mix  in triplicate. PCR and data collection were performed on a M×3000P™ Real-Time PCR system .μg) was resolved on a 10 to 12% denaturing polyacrylamide gel and transferred electrophoretically to a nitrocellulose membrane . After incubation with primary antibodies at 4°C overnight, the immunoreactive proteins were localized with horseradish peroxidase-conjugated secondary antibodies . The reactants were developed using an enhanced chemiluminescence kit (Amersham Biosciences).The protein content of each lysate was determined by a Bio-Rad protein assay . Each lysate  was cloned into the pGL3-Basic vector  to be used as a reporter assay. The AsSn (or pIRES2-EGFP vector) and fly-luciferase plasmids were cotransfected into MDA-MB-231 and MDA-MB-435S cells using Lipofectamine 2000 (Invitrogen). After 48 h, firefly and Renilla luciferase activities were measured using the Dual Luciferase Reporter Assay kit (Promega).μl pipette tip was used to create a scratch down the middle of each well. Representative images were photographed using phase-contrast microscopy at the indicated times.For the wound-healing migration assay, ∼250,000 cells were seeded in six-well plates and grown to 100% confluence. A 10-4 cells were seeded on the top of Transwell membranes treated with Matrigel matrix  following the manufacturer’s instructions. After 10 h of incubation, filters were fixed with 4% paraformaldehyde ; the number of cells on the top surface of the filters was estimated by counting three independent visual fields using a microscope. The average number of cells in four replicate wells was determined for each cell line in each of three independent experiments.For the cell migration assay, 1×103) were plated in 96-well plates and allowed to attach and grow in regular medium for 4 h. At each time point , 10 μl MTT (5 mg/ml) was added to each well. The reaction was stopped after 4 h of incubation by adding 150 μl dimethyl sulfoxide (DMSO). The optical density (OD) value was obtained by measuring the absorbance at a wavelength of 570 nm. The proliferation assay was performed in triplicate and repeated three times.Cells , which were incubated with antibodies to RhoA, Rac1 or Cdc42 (diluted 1:1000).The activities of Rac1, Cdc42 and RhoA were studied as described previously . GlutathFemale athymic nude mice (BALB/c nu/nu) aged between four and five weeks were obtained from the Shanghai Institute of Medical Material . Parental and AsSn-transfected MDA-MB-231 and MDA-MB-435s cells were harvested, washed, resuspended in PBS and injected into the mammary fat pad of mice. Tumor growth was measured three times each week. For survival analysis, the mice were euthanized and sacrificed when they appeared moribund. All experiments were performed at least twice, and samples from the breast tumors, lungs, liver and lymph nodes were obtained. Tissues for histological examination were fixed and embedded in paraffin using standard methods.Spearman’s rank tests were used to evaluate the correlation between Snail or RhoA expression and clinical pathological parameters. SPSS 16.0 software  was used to carry out all statistical analyses. P<0.05 was considered significant to indicate a statistically significant difference.The expression and subcellular localization of Snail and RhoA were studied in a set of specimens derived from 20 normal breast tissues and 60 breast cancer tissues. In the normal breast tissues examined, only faint nuclear staining for Snail and cytoplasmic staining for RhoA were detected, whereas the breast carcinomas exhibited strong staining for Snail and RhoA . To furtAsSn was stably introduced into the human breast cancer cell lines MDA-MB-231 and MDA-MB-435S, which express relatively high levels of Snail mRNA and exhibit the invasive phenotype. A low level of Snail expression in AsSn MDA-MB-231 clones was demonstrated by western blotting with anti-Snail antibody, while Snail expression was not affected in the mock-transfected cells. A similar result was observed in the MDA-MB-435S cell line , demonstWhether the introduction of AsSn affected the expression of EMT genes using western blotting analysis MDA-MB-231 was investigated. Analysis of several independent clones revealed increased expression levels of E-cadherin and a significant decrease in endogenous fibronectin and MMP-2 expression in AsSn cells, whereas RhoA expression was reduced by ∼60% in AsSn cells .To exclude the possibility that the knockdown by the anti-sense vector was not Snail-specific, the mRNA expression level of Slug, a close homolog of Snail, was detected by real-time PCR. No significant changes in the level of endogenous Slug mRNA were detected in the AsSn- or mock-transfected clones .Furthermore, the mock-transfected clones exhibited spindle-shaped cells and fibroblastic morphology, whereas AsSn clones typically displayed a cobblestone-like, epithelial morphology .It was observed that the downregulation of Snail in breast tumor cells significantly reduced cell migration from the edge of the wound 24 h after scratching . Similarin vitro.To exclude the possibility that differences in the growth rates of the mock-transfected and AsSn cells affected the interpretation of these results, cell growth was observed over 72 h and curves were plotted. There was no significant difference between the proliferation of the mock-transfected and AsSn cells after 72 h, excluding such a possibility . This reA reporter construct containing the RhoA promoter was transfected into the same cells and which were analyzed for promoter activity. The transient transfection results showed a two- to three-fold repression of RhoA promoter activity by Snail downregulation, suggesting that RhoA expression was under the control of Snail .To investigate whether AsSn was capable of inactivating Rho GTPase and whether this inactivation was involved in the decreased migration ability, GTPase assays were performed using GST fusion proteins with binding domains that bind only activated forms of these GTPases. As shown in in vitro. A significant decrease in the invasion ability of AsSn cells was obtained with transfectants expressing DN-RhoA, whereas overexpression of Act-RhoA increased the invasion ability of the transfectants compared with transfectants expressing GFP alone  or active (V14-RhoA) RhoA vectors were transfected into the cells and the invasion ability was measured FP alone . WesternFP alone . WhetherMice that received an injection of mock-transfected cells developed tumors of >1.5 cm in diameter  and theiWestern blotting analysis showed decreased levels of Snail and RhoA proteins in the tumors that received an injection of AsSn cells , comparein vitro and in vivo metastasis properties of estrogen receptor (ER)-negative MDA-MB-231 and MDA-MB-435S cell lines transfected with AsSn and DN-RhoA or Act-RhoA vectors.In the present study, it was observed that Snail and RhoA were coexpressed at significantly higher levels in breast cancer tissues compared with normal tissues, according to immunohistochemical analyses and western blotting. Analysis of the clinicopathological characteristics  shows foBy inhibiting Snail expression, clear changes were observed in the expression of several important components of the EMT proteome in AsSn cells. The EMT proteome reflects a fundamental change in the proteins gained or lost in the transition of tumor epithelia to metastatic cells . Since RCell invasion is associated not only with the ability to be motile, but also the ability to degrade the extracellular matrix. The decreased invasion ability of AsSn cells was coupled with the downregulated expression of RhoA and MMP-2, suggesting that cell motility and extracellular matrix degradation are likely to be functionally interdependent for cell invasion. We suggest that Snail may modulate RhoA expression and trigger Rho GTPase-dependent signaling, leading to the control of MMP-2 expression.in vivo. At the end of the experiment, a significant reduction in the volume of tumors induced by AsSn cells and less lymph node metastasis was detected compared with mock-transfected cells. The results indicate that the full biological effect of blocking Snail must be markedly affected by the tumor microenvironment. Furthermore, mice injected with AsSn cells survived longer than those injected with mock-transfected cells. Significantly decreased levels of RhoA protein were observed in the tumors derived from mice injected with AsSn cells. These details clarify the role of Snail in the regulation of RhoA, which is correlated with tumor metastasis by affecting cell movement.The metastasis and survival time after injection of mock-transfected and AsSn MDA-MB-231 cells into BALB/C SCID mice was further tested A previous study indicated that the use of RNA interference may be an effective tool for blocking Snail function . We havein vivo. Based on these findings, Snail may be considered, in the future, as a putative molecular target for antineoplastic therapy.In conclusion, the present data support a novel role for Snail in the progression of breast tumors and provide evidence that this effect is mainly mediated through the regulation of RhoA activity which is involved in cell movement and growth


Contrast-induced AKI (CI-AKI) has been one of the leading causes for hospital-acquired AKI and is associated with independent risk for adverse clinical outcomes including morbidity and mortality. The aim of this review is to provide a brief summary of the studies that focus on nonpharmacological strategies to prevent CI-AKI, including routine identification of at-risk patients, use of appropriate hydration regimens, withdrawal of nephrotoxic drugs, selection of low-osmolar contrast media or isoosmolar contrast media, and using the minimum volume of contrast media as possible. There is no need to schedule dialysis in relation to injection of contrast media or injection of contrast agent in relation to dialysis program. Hemodialysis cannot protect the poorly functioning kidney against CI-AKI. P < 0.001) or diabetes mellitus  were at increased risk for the development of CI-AKI . . 18]. Electronic Warning Systems. Cho et al. [2, the physician was immediately alerted by a warning message to consider prophylactic measures for CI-AKI. This electronic warning systems significantly decreased the risk of CI-AKI  [o et al.  demonstr = 0.02) .Prior to contrast media exposure, the use of established nephrotoxic drugs, for example, cyclosporine A, aminoglycoside, and NSAID including COX-2 inhibitors, should be stopped for at least 2 days. Diabetic patients with preexisting renal impairment should withhold metformin for 48 hours because lactic acidosis may occur once CI-AKI develops. However, patients with normal renal function who are taking metformin are not at risk of CI-AKI and should be assessed according to their overall clinical conditions .The pathogenic role of angiotensin-converting enzyme (ACE) inhibitors in CI-AKI is still controversial. Some studies pointed out that ACE inhibitors were effective in the prevention of CI-AKI, while some concluded that they were associated with increased risk of CI-AKI, especially for patients with preexisting renal impairment. On one hand, experimental data suggest that activated renin-angiotensin-aldosterone system, enhanced endothelin-1, and increased ROS play important roles in the pathogenesis of CI-AKI and these can be inhibited by ACE inhibitors. On the other hand, ACE inhibitors impair angiotensin II synthesis. In the presence of contrast-induced acute reduction of renal blood flow, blunting the vasoconstriction of efferent arterioles by decreased angiotensin II levels may have a deleterious effect on GFR via decreasing the intraglomerular pressure. These opposite effects probably explain why the studies to date have provided inconclusive results on this issue .Kwok et al.  performeLapi et al.  reportedAll patients receiving contrast media should have an optimal volume status at the time of exposure. Indeed, volume supplementation plays an important role in the prevention of CI-AKI via two mechanisms. First, expansion of the intravascular space is thought to blunt the vasoconstrictive effect of contrast on the renal medulla via suppression of vasopressin secretion, inhibition of the renin-angiotensin axis, and increased synthesis of vasodilatory renal prostaglandins. Second, intravenous fluid replacement is believed to attenuate the direct toxic effect of contrast agents on tubular epithelial cells by decreasing the concentration and viscosity of contrast media in the tubular lumen. This attenuating effect is the result of volume-mediated inhibition of proximal tubular salt and water reabsorption and decreasing contact time from the associated increase in tubular flow .3) infusion may not only allow for shorter period of volume supplementation but also further reduce the generation of injurious ROS. Typically, patients should receive 154 mEq/L of NaHCO3, as a bolus of 3 mL/kg/hour for 1 hour prior to contrast media administration, followed by an infusion of 1 mL/kg/hour for 6 hours after the procedure [In patients without heart failure, parenteral isotonic normal saline (0.9% NaCl) without any diuretics should be started 12 hours prior to contrast media administration with an infusion rate of 1 mL/kg/hour and continued for 24 hours. The use of sodium bicarbonate  compared with IOCM  [3  [3 .P = 0.73). In addition, Marenzi et al. [Surprisingly, a recent meta-analysis  reportedi et al.  also demIn a meta-analysis of 25 trials, the pooled odds ratio of CI-AKI with LOCM was 0.61 , 0.48–0.77) times that after high osmolar contrast media (HOCM). For CKD patients, this odds ratio was 0.5 , while it was 0.75  in patients without prior renal insufficiency. These data suggest that HOCM is generally more nephrotoxic than LOCM and the use of LOCM may be beneficial, particularly in CKD patients . FurtherHowever, the risk of CI-AKI with two LOCM including iohexol and ioxaglate was significantly higher than other LOCM  and the IOCM (iodixanol) in many studies , 36. In Hence, the CI-AKI Consensus Working Panel suggests that, for intra-arterial administration in high-risk patients with CKD, particularly those with diabetes mellitus, nonionic IOCM are associated with the lowest risk of CI-AKI . In addiThe contrast media should be prewarmed to 37°C for decreasing viscosity .P = 0.01) with intra-arterial route, but not with intravenous application , when compared with a pool of LOCM in a recent meta-analysis [In a previous study, the incidences of AKI after intra-arterial and intravenous contrast media administrations were comparable ; howeveranalysis .Multivariate analyses have established the correlation between higher dose of contrast media and risk for CI-AKI . The CI-Tan et al.  proposedAutomated Contrast Injector Systems (ACIS). Contrast volume is a major modifiable risk factor for CI-AKI. ACIS are believed to be associated with a reduction in the total volume of contrast media. Unfortunately, the use of ACIS was associated with a statistically significant lower in the average volume of contrast media with no difference in the incidence of CI-AKI or new need for dialysis [dialysis .P < 0.001, 95% CI −54 to −35). The CI-AKI incidence was significantly reduced by 15% with an odds ratio of 0.85  for those using ACIS compared with manual injection [In a recent meta-analysis in 79,694 patients from 10 studies, ACIS reduced contrast volume delivery by 45 mL/case  can effectively remove 60–90% of contrast media from the blood. Because most contrast media are middle-sized molecules, the main factors potentially affecting CI-AKI depend on HD efficacy of contrast media removal. Blood flow, membrane surface area, molecular size, transmembrane pressure, and dialysis time are important factors that contribute to the efficacy of HD . GeneralPeritoneal dialysis is also effective in removing contrast agents from the body but takes longer duration time period than HD. Three weeks of continuous ambulatory peritoneal dialysis is needed for completely removing the agent. Contrast media can be removed effectively by various peritoneal dialysis modalities, including intermittent peritoneal dialysis, automated peritoneal dialysis, and continuous ambulatory peritoneal dialysis .A previous meta-analysis  and a re2. Approximately, 60% had diabetes mellitus and the mean volume of total contrast media (iohexol) was more than 100 mL that contributed to a high risk of CI-AKI. All patients were given intravenous normal saline at 1 mL/kg/hour for 6 hours before and 12 hours after contrast media exposure. HD was prescribed by using a high-flux dialyzer. The blood flow was 150 mL/min, the duration of dialysis was 4 hours, and the dialysate flow was 500 mL/min. To lessen the hemodynamic changes, 200 mL normal saline priming was administered before dialysis and no fluid removal was prescribed in the dialysis group. HD was initiated at an interval of 81 ± 32 min, ranging from 45 to 180 min, after exposure to the contrast media. A potential important limitation of this study is the use of serum creatinine to diagnose CI-AKI. Indeed, any initial reduction of serum creatinine in HD group is likely a falsely beneficial effect resulting from creatinine removal. The bicarbonate dialysate used and additional fluid replacement might be another important effect. However, the prophylactic dialysis seems not to be important even in advanced CKD.Interestingly, Lee et al.  demonstrIn addition, the risks of dialysis procedures and the much greater cost should be considered. As such, the Contrast Media Safety Committee of ESUR states that there is no need to schedule the dialysis in relation to the injection of contrast media or the injection of contrast agent in relation to the dialysis program. Hemodialysis does not protect the poorly functioning kidney against CI-AKI .As a strong relationship between contrast load and incidence of CIN is obviously demonstrated, alternative strategies to limit the systemic contrast exposure are being developed. The CINCOR catheter  has been innovated for removing contrast media from coronary sinus shortly after contrast delivery . Contrasn = 174) or to a control group . Continuous infusion of isotonic saline solution (1 mL/kg/hour) was administered 12 hours before the procedure until 12 hours after the procedure in both groups. The PaO2 at the baseline was significantly higher in the oxygen preconditioning group than the control group . The authors demonstrated that the oxygen preconditioning reduced the incidence of CI-AKI, particularly in CKD patients, via decreased intrarenal hypoxia. Therefore, this simple strategy to attenuate CI-AKI might be beneficial in CKD patients.Sekiguchi et al.  randomlyP = 0.002). The IPC was performed as 4 cycles of alternating 5-minute inflation and 5-minute deflation of a standard upper-arm blood pressure cuff to the individual's systolic blood pressure plus 50 mm Hg to induce transient and repetitive arm ischemia and reperfusion. Although the protective mechanism of IPC is still unestablished, it is postulated that a remote organ might release humoral factors such as adenosine, bradykinin, or erythropoietin into the systemic circulation, all of which subsequently protect the remote region or organ.Er et al.  demonstr2, diabetes mellitus, recent nephrotoxic exposure, and intra-arterial route. In at-risk patients, consider an alternative imaging method, start volume expansion, and utilize the lowest dose of contrast media consistent with a diagnostic result. Finally, determining eGFR 48–72 hours after receiving contrast media should be performed for CI-AKI detection.The Contrast Media Safety Committee of ESUR  has updaSimilarly, the KDIGO Clinical Practice Guidelines on Acute Kidney Injury  recommenIn conclusion, contrast-induced AKI (CI-AKI) has been one of the leading causes for hospital-acquired AKI and is associated with independent risk for adverse clinical outcomes including morbidity and mortality. To prevent CI-AKI in patients who are receiving contrast media, every effort is required, including routine identification of at-risk patients, the use of appropriate hydration regimens, withdrawal of nephrotoxic drugs, selection of LOCM or IOCM, and using the minimum volume of contrast media as possible. There is no need to schedule the dialysis in relation to the injection of contrast media or the injection of contrast agent in relation to the dialysis program. Hemodialysis does not protect the poorly functioning kidney against CI-AKI.


Previous studies have demonstrated that structural deficits and functional connectivity imbalances might underlie the pathophysiology of obsessive-compulsive disorder (OCD). The purpose of the present study was to investigate gray matter deficits and abnormal resting-state networks in patients with OCD and further investigate the association between the anatomic and functional alterations and clinical symptoms.Participants were 33 treatment-naïve OCD patients and 33 matched healthy controls. Voxel-based morphometry was used to investigate the regions with gray matter abnormalities and resting-state functional connectivity analysis was further conducted between each gray matter abnormal region and the remaining voxels in the brain.Compared with healthy controls, patients with OCD showed significantly increased gray matter volume in the left caudate, left thalamus, and posterior cingulate cortex, as well as decreased gray matter volume in the bilateral medial orbitofrontal cortex, left anterior cingulate cortex, and left inferior frontal gyrus. By using the above morphologic deficits areas as seed regions, functional connectivity analysis found abnormal functional integration in the cortical-striatum-thalamic-cortical (CSTC) circuits and default mode network. Subsequent correlation analyses revealed that morphologic deficits in the left thalamus and increased functional connectivity within the CSTC circuits positively correlated with the total Y-BOCS score.This study provides evidence that morphologic and functional alterations are seen in CSTC circuits and default mode network in treatment-naïve OCD patients. The association between symptom severity and the CSTC circuits suggests that anatomic and functional alterations in CSTC circuits are especially important in the pathophysiology of OCD.  Obsessive-compulsive disorder (OCD), a common and disabling neuropsychiatric disorder, affects 2%–3% of the general population . OCD is Previous neuropsychological and animal studies have shown that abnormalities of cortical-striatal-thalamic-cortical (CSTC) circuits may play a key role in the pathogenesis of OCD -5. AlthoThere are increasing functional studies which have provided strong evidence that the pathophysiology of OCD is unlikely to be the result of a single abnormal brain region or neurotransmitter system. Instead, it could be conceptualized as a distributed neuronal brain network ,21. TherIn the current study, we combined voxel-based morphometry (VBM) and resting-state functional connectivity analysis in order to perform a comprehensive evaluation of the neural circuitry of OCD. Along these lines, the main objective of this study was to (1) identify brain regions with gray matter abnormalities in first-episode treatment-naïve patients with OCD, (2) investigate the brain functional connectivity using the observed gray matter abnormalities areas as seed regions, and (3) explore the clinical significance of structural deficits and functional connectivity by focusing on their association with symptom severity and disease duration in OCD patients.The medical ethics committee of Third Military Medical University  approved the current study and all participants gave written informed consent to the participation according to the Declaration of Helsinki. Thirty-four patients who were seeing a doctor for the first time and met the DSM-IV criteria for OCD were recruited for this study from the Department of Clinical Psychology of the Southwest Hospital between September 2010 and December 2012. Diagnosis of OCD and duration of illness were determined by two qualified psychiatrists (Dr W. Qu and Dr J. Guo) using the Structured Clinical Interview according to the DSM-IV criteria (SCID). The severity of OCD symptoms was assessed using the Yale-Brown Obsessive-Compulsive Scale (Y-BOCS) ,37. In aThirty-four healthy controls were recruited among hospital staff and by advertisements on the internet. All controls were screened by using the SCID-Nonpatient Edition to ascertain that there was no personal history of psychiatric and neurologic illnesses. The controls were also interviewed to confirm the lifetime absence of psychiatric diseases in their first-degree relatives. Exclusion criteria for OCD patients and healthy controls were as follows: a history of other psychiatric or neurological illness; a history of drug or alcohol abuse; pregnancy; serious physical illness; and contraindications to MR scanning. Conventional MR images (T2-weighted images) were inspected by two experienced neuroradiologists (Dr J. Wang and Dr H. Li) to exclude gross abnormalities. Co-morbid depressive and anxious symptoms were not considered as an exclusion criterion, if OCD was the primary clinical diagnosis. Of the original participants, one female patient and one male healthy control were excluded from the final analysis; the patient was excluded because of excessive head motion during MR scanning (>2 mm in translation) and the control was excluded owing to an incidental finding on conventional MR images . The final study consisted of 33 OCD patients and 33 healthy controls. The two groups were well-matched for age, gender, and years of education. All participants were right-handed. Clinical and demographic data from all 66 participants are shown in 2; matrix, 64 × 64; and isotropic voxel, 4 × 4 × 4 mm3. For each participant, the fMRI scanning lasted for 480 s and 240 volumes were obtained. In addition, a high resolution structural T1-weighted anatomic sequence was acquired in a sagittal orientation using a 3-dimensional magnetization-prepared rapid gradient-echo (3D MP-RAGE) sequence, as follows: repetition time, 1900 ms; echo time, 2.52 ms; flip angle, 15°; slice thickness, 1 mm; matrix, 256 × 256; and isotropic voxel, 1 × 1 × 1 mm3.All structural and functional images were obtained using a 3.0 T MRI system  with an eight-channel phased array head coil. During the MRI scans, all participants were instructed to remain relaxed with their eyes closed and lie still without moving. The resting-state functional images were acquired using an echo-planar-imaging (EPI) sequence: 36 axial slices with a slice thickness of 4 mm and no slice gap; repetition time, 2000 ms; echo time, 30 ms; flip angle, 90°; field of view, 256 × 256 mmThe voxel-based morphometric analysis was performed using SPM8 software  implemented in MATLAB 2010b . First, all structural MR images were manually reoriented to place the anterior commissure at the origin of the 3D MNI space. Then, the structural images were segmented into gray matter, white matter and cerebrospinal fluid using the unified standard segmentation option in SPM8. After segmentation, the gray matter template for a group of individuals was generated from the entire image dataset using the Diffeomorphic Anatomical Registration Through Exponentiated Lie algebra (DARTEL) toolbox following John Ashburner’s chapter in the standard version . Next, tt-tests. Age, gender, and total intracranial volume were modeled as covariates of no interest. The statistical significance of group differences in each region was set at a p<0.05 with family-wise error correction. To identify the association between gray matter abnormalities and clinical characteristics including Y-BOCS score and disease duration, the average values of gray matter volume for all the voxels in abnormal areas revealed by VBM analysis, were extracted and correlated with the Y-BOCS score and disease duration using Pearson correlation analysis.Voxel-wise comparisons of gray matter volume between the OCD and control groups were performed using two-sample Functional connectivity preprocessing and statistical analysis were also done with SPM8 software. To avoid manipulation error confounds and standardize the process, we used the batch-processing tool data processing assistant for resting-state fMRI  . For ther values were converted using Fisher’s r-to-z transformation to improve the Gaussianity of their distribution.Functional connectivity was analyzed using the REST software package (http://www.restfmri.net) using a seed voxel correlation approach . Becauset-tests were used. Age, gender, years of education and total gray matter volume were entered as co-variates of no interest. The significance level of group differences was set at a p<0.05 with multiple comparison corrected by AlphaSim methods (combined height threshold of a p<0.001 and a minimum cluster size of 22 voxels). Subsequently, the group differences were correlated with the total Y-BOCS score and disease duration using Pearson correlation analysis.To compare the difference in functional connectivity between the OCD and healthy control group, two-sample To further evaluate the reproducibility of our findings, we conducted the following procedures.In the preprocessing of resting state fMRI data, there is much debate in the community about the use of global signal regression -45. AlthPrevious studies have shown that that resting-state brain function may be affected by structural gray matter alteration . In thisp = 0.61) in the total gray matter volume between first-episode OCD patients (609.98±64.32 ml) and healthy controls (615.63±60.95 ml). Compared with healthy controls, OCD patients showed significantly increased regional gray matter volume mainly in the left caudate , left thalamus  and posterior cingulate cortex , as well as decreased gray matter volume in the bilateral medial OFC , left ACC  and left inferior frontal gyrus . To characterize the alteration of functional networks involving brain areas associated with abnormal gray matter volume, functional connectivity analysis was performed using a seed voxel correlation approach. According to the morphometric analysis results, six brain regions which showed abnormal gray matter volume in OCD patients were selected as seed regions for functional connectivity analysis on resting-state fMRI data; specifically, 3 increased gray matter volume areas  and 3 decreased gray matter volume areas  were selected. There were no significant differences between the two groups in head motion , cerebellum, and left caudate, as well as decreased functional connectivity in the right inferior parietal lobe. When the seed was located in the PCC, patients with OCD showed increased functional connectivity in the cerebellum, right medial frontal gyrus, left thalamus, and bilateral middle temporal gyrus. When the seed was located in the medial OFC, patients with OCD showed increased functional connectivity in the bilateral caudate and left ACC, as well as decreased functional connectivity in the bilateral lateral OFC, inferior temporal gyrus, left middle frontal gyrus and left angular. When the seed was located in the left ACC, patients with OCD showed increased functional connectivity in the left caudate, right ACC and medical OFC, as well as decreased functional connectivity in the right inferior temporal gyrus, cerebellum, and left angular. When the seed was located in the left inferior frontal gyrus, patients with OCD showed increased functional connectivity in the left putamen, caudate, and cerebellum, as well as decreased functional connectivity in the left superior temporal gyrus , Table 2r = 0.68, p<0.001). In addition, significant positive correlations were also found between the total Y-BOCS scores and the strength of the functional connectivity of multiple areas within the cortical-striatum-thalamic circuits. When the seed was located in the left caudate, significant positive correlations were observed between the total Y-BOCS scores and the strength of functional connectivity in the bilateral lateral OFC  and right putamen . When the seed was located in the medial OFC, significant positive correlations were observed between the total Y-BOCS scores and the strength of functional connectivity in the bilateral caudate . Moreover, by using the above morphologic deficits areas as seed regions, functional connectivity analysis revealed abnormal functional integration in the CSTC circuits and default mode network in OCD patients. Subsequent correlation analyses revealed that morphologic deficits in the left thalamus and increased functional connectivity within the cortical-striatum-thalamic circuits positively correlated with the total Y-BOCS score in patients with OCD, providing a link between CSTC circuits connectivity and OCD clinical symptom severity. In recent years, many neuroimaging studies have explored the pathophysiology of OCD. These studies have contributed greatly to the development of neurocircuitry models of OCD, which emphasize the dysfunction of CSTC circuits in OCD patients ,24,26,27In neuroimaging study on OCD, increasing attentions have now been paid to the DMN. The DMN is a prominent large-scale brain network characterized by a deactivation during goal-directed cognitive performance and increased activity in self-referential processing ,67. The There is increasing evidence that disrupted functional connectivity in OCD is considered to be a potential systems-level substrate of this disease. Functional MRI studies have reported alterations of functional connectivity in OCD patients and offered a series of meaningful information about the pathogenesis of OCD. Harrison et al.  used dorIn the present study, using gray matter deficit areas as seed regions in a cohort of treatment-naïve OCD patients, we found altered connectivity mainly in the CSTC circuits, DMN, and some other regions, including the cerebellum and temporal cortex. Relative to healthy controls, patients with OCD showed significantly increased connectivity within the CSTC circuits that included the bilateral lateral OFC, medial OFC, ACC, caudate, putamen thalamus, and left inferior frontal gyrus. We speculate that the OCD patients perhaps need to enhance the functional connectivity of these regions to compensate for their difficulty processing the alternating stimulus context, consistent with their phenomenological difficulty processing changing environmental contingencies and their overall cognitive inflexibility . Most prIn the current study, we also revealed that the strength of functional connectivity within CSTC circuits was positively correlated with the total Y-BOCS scores of OCD patients, including the bilateral caudate, bilateral lateral OFC, medial OFC and right putamen. Previous neuroimaging studies reported that the metabolism in OFC of patients with OCD could predict the total Y-BOCS scores of patients with OCD ,79. HarrSeveral limitations should be considered when interpreting the results of the present study. First, the data are cross-sectional, therefore, the dynamic changes of brain structure and function during the progression of OCD could not be revealed by the current study. Future longitudinal studies can reveal deeper insight into the pathophysiology of OCD. Second, the OCD patients in the current study were heterogeneous in major symptom dimensions, and several structural and functional studies ,26 showeIn conclusion, our findings provides evidence that morphologic and functional deficits are seen in CSTC circuits and default mode network in treatment-naïve OCD patients. At the same time, findings from the present study, along with future studies clarifying the causes of the morphologic and functional changes reported, may provide new insight into the underlying pathophysiology of OCD.Figure S1Within-group functional connectivity maps of the healthy controls and OCD patients with and without global signal regression. Maps threshold were set at p<0.05 with AlphaSim correction.(TIF)Click here for additional data file.Figure S2Between-group differences in functional connectivity without global signal regression. Maps threshold were set at p<0.05 with AlphaSim correction. (TIF)Click here for additional data file.Figure S3Between-group differences in functional connectivity with correcting gray matter volume and global signal regression. Maps threshold were set at p<0.05 with AlphaSim correction.(TIF)Click here for additional data file.