Human papillomavirus (HPV) infection is a causative factor for various cancers of the anogenital region and oropharynx, and is supposed to play an important cofactor role for skin carcinogenesis. Evasion from immunosurveillance favors viral persistence. However, there is evidence that the mere presence of oncogenic HPV is not sufficient for malignant progression and that additional tumor-promoting steps are required. Recent studies have demonstrated that HPV-transformed cells actively promote chronic stromal inflammation and conspire with cells in the local microenvironment to promote carcinogenesis. This review highlights the complex interplay between HPV-infected cells and the local immune microenvironment during oncogenic HPV infection, persistence, and malignant progression, and discusses new prospects for diagnosis and immunotherapy of HPV-associated cancers. Approximately 15–20% of all cancers are caused by infectious agents . The cauInvasive cancer is not an immediate consequence of HPV infection. HPV-induced carcinogenesis takes years or decades to occur, and there is increasing evidence that additional tumor-promoting steps are required . It is wWhile screening programs have greatly reduced the burden of cervical cancer in developed countries, current diagnostic tests cannot discriminate between lesions that will progress to invasiveness and those that do not. This results in an overtreatment of high-grade lesions that are detected during screening . A betteThis review focuses on the current understanding of the complex interplay between HPV-infected cells and the local immune microenvironment during HPV infection and HPV-associated carcinogenesis, and discusses novel prospects for diagnosis and immunotherapy.Human papillomaviruses are non-enveloped double-stranded (ds) DNA-viruses that are transmitted sexually or by smear infection . More thGenus β-HPVs have been implicated in ultraviolet (UV) light-induced non-melanoma skin cancer in patients suffering from the inherited disease Epidermodysplasia verruciformis  and a co+ T cell-mediated responses directed against viral early proteins [In mucosal as well as cutaneous HPV-associated carcinogenesis it is well accepted that the immune system has an important surveillance function. In immunocompetent individuals, up to 90% of anogenital HPV infections are cleared within two years ,37,38 anproteins ,40. Convproteins ,44,45,46HPV infections that escape immune control can persist and a certain proportion progresses to cancer. Recent studies have shed light on the immune system as a double-edged sword in HPV-associated carcinogenesis and evidence is increasing that the role of the immune system changes in a stage-dependent manner. At earlier stages, anti-viral immunity predominates and the virus has adopted strategies to counteract immunosurveillance in order to establish persistence in the epithelium. However, at later stages of the disease, HPV-transformed cells reprogram the local immune microenvironment and rather initiate chronic stromal inflammation, which then serves to promote progression of precursor lesions to invasive cancer .To maintain a first line of defense against infections agents, skin and mucosal surfaces are equipped with efficient immune sentinels and immune effector mechanisms ,48. KeraFor productive infection HPV depends on the keratinocyte differentiation program. After having entered the proliferating basal keratinocytes, HPV gene expression is low and vegetative replication dramatically increases only in the more differentiated layers of the epithelium that are bound to desquamate shortly. The minor levels of protein expression in the lower epithelial layers, the well-directed non-cytolytic genome amplification restricted to the differentiated layers, and the lack of a viremic phase in the viral life cycle are thought to passively help the virus escape immune recognition ,53. ThusAlthough HPVs encode only a limited number of regulatory genes, they engage various active strategies to counteract immune recognition and cell-autonomous immune responses at different levels . MucosalIn contrast to RNA- and other dsDNA-viruses like herpes-simplex or vaccinia viruses that activate the inflammasome in keratinocytes , mucosalIn contrast to IL-1β, IL-1α, an intracellularly stored alarmin, is detectable throughout cervical carcinogenesis . IL-1α aNotably, oncolytic viruses are currently under investigation for cancer therapy and it has become clear that immune activation is an important part of their anti-tumor activity . Whether+ T cells [Professional antigen-presenting cells connect innate and adaptive immunity. APCs migrate to secondary lymphatic tissues where they encounter specific T cells, which subsequently become activated and are redirected to the sites of infection. Local factors released from epithelial cells critically influence recruitment, differentiation and activation of APCs. Although the role of murine Langerhans cells has been controversially discussed, human Langerhans cells have been shown to prime and cross-prime naive CD8 T cells , which a+ Langerhans cell precursors [Evidence is emerging that HPV infection actively interferes with human Langerhans cell homeostasis in the epidermal compartment. Both cutaneous and mucosal HPV-infected epithelia harbor only low numbers of Langerhans cells ,83 and tecursors . In normecursors .Thus, HPV infection actively suppresses cell-autonomous viral recognition and acute inflammatory signaling in the host keratinocyte as well as recruitment of epithelial APCs. The low levels of inflammatory cytokines produced by HPV-infected cells may further contribute to the lack of APC activation, eventually allowing the virus to escape from local immunosurveillance and to persist in the epithelium.There is ample evidence that oncogenic HPV infection in the human cervix and skin starts with expansion of the epithelial stem cell compartment ,87, whicHowever, with increasing dysplasia a dramatic increment of stromal infiltration with immune cells is noted in cervical patient biopsies ,13,83,912+-signaling. MMP-9 has been detected in monocytes starting to infiltrate cervical high-grade lesions at the switch to malignancy [A clue came from the observation that HPV-transformed cells potently up-regulate chemoattractants in the tumor stroma ,12. In mlignancy . Local plignancy . Transgelignancy ,99. Impolignancy .Neutralization experiments revealed that HPV-transformed cells induce CCL2 and subsequent MMP-9 expression in monocytes via a combination of IL-6 and macrophage colony-stimulating factor (M-CSF). They activate the janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) signaling pathway in monocytes and various JAK/STAT3-inhibitors are able to interfere with this pro-tumorigenic response .During productive infection HPV suppresses IL-6 similar to other cytokines . However+ cytotoxic T cell responses. Under normal conditions, the migration receptor CCR7 becomes expressed on their surface during maturation, ensuring their responsiveness to lymph node homing chemokines [+ dendritic cells are present in the tumor stroma. However, they were found to be largely devoid of CCR7 expression [+ phenotypically mature dendritic cells. As a consequence, expression of the chemokine receptor CCR7 is suppressed in the dendritic cells and their migration towards lymph node homing chemokine is blocked [Stromal myelomonocytic cells can either differentiate into dendritic cells, APCs destined to mount adaptive immune responses, or into macrophages, tissue-resident phagocytes. For the initiation of adaptive immunity, dendritic cells have to mature as indicated by surface expression of CD83, to up-regulate major histocompatibility complex class I and II required for antigen presentation as well as co-stimulatory molecules such as CD80 and CD86 required for T-cell activation, and finally to produce cytokines that polarize T helper 1 (Th1) cells required for efficient CD8emokines ,105. A semokines ,107. In pression . As the  blocked . This ma blocked ,109.In contrast to CCR7, MMP-9 is potently up-regulated in immature and mature dendritic cells. Notably, both CCR7 and MMP-9 up-regulation are mediated by IL-6 from the cervical cancer cells ,110. ThuIn cervical cancer stroma M2-polarized macrophages accumulate with low IFN-γ production and a low capacity to stimulate T cell proliferation . M2 macr+ T cells in cervical cancer [Besides neoplastic cells, M2-polarized macrophages were also found to express the programmed death-ligand 1 (PD-L1) . There il cancer  suggestil cancer ,116. Notl cancer .clinicaltrials.gov).From these studies evidence is increasing that paracrine IL-6 as well as subsequent STAT3 activation and NF-κB suppression are central for reprogramming myelomonocytic cells creating a pro-tumorigenic and immunosuppressive microenvironment in cervical carcinogenesis. As a consequence, phenotypically mature but functionally impaired dendritic cells and macrophages are actively retained in the tumor stroma and this may (1) prevent the initiation of anti-tumoral adaptive Th1 immune responses; (2) suppress cytotoxic T cell activity; and (3) contribute to an aberrant local expression of MMP-9 that promotes tumor growth and vasculogenesis. This suggests that the IL-6/JAK/STAT3 signaling pathway might be an interesting target to revert immune deviation in cervical cancer. In fact, the IL-6/JAK/STAT3 signaling pathway is “druggable” at various levels  and clin+ T cell lineage that can exert either regulatory or inflammatory functions [In human biopsies of cervical carcinogenesis, enhanced infiltration of Th17 cells is observed with increasing stages of disease and this correlates with up-regulation of CCL20 expression in the stromal mesenchymal compartment . It is wunctions . In variunctions ,123,124.unctions . Recentlunctions . ThorougThese studies provided evidence that paracrine IL-6 shapes a proinflammatory and immunosuppressive microenvironment in cervical carcinogenesis via activation of two pathways in stromal immune and stromal mesenchymal cells, the JAK/STAT3 and the C/EBPβ signaling pathway. In HPV-driven carcinogenesis, IL-6-induced JAK/STAT3- and C/EBPβ-driven stromal inflammation may play a key role in promoting tumor progression. At the same time paracrine IL-6 can limit NF-κB-dependent anti-viral and anti-tumor immune responses in APCs, further highlighting IL-6 as an attractive target for adjuvant immunotherapy of cervical cancer.In HPV-driven carcinogenesis STAT3 is not only activated in the tumor microenvironment. Also, epithelial cells in cervical high-grade lesions display strong pTyr705-STAT3 activation by far exceeding activation levels in normal exocervical epithelium or low-grade lesions ,126. StuNotably, in invasive cervical cancers STAT3 activation is often retained at the tumor margin adjacent to the stroma, while in other parts of the tumor STAT3 activation declines compared to cervical high-grade lesions . This suStrikingly, when the IL-6/STAT3-signaling pathway is activated in cervical cancer cells, they are more efficiently killed by chemotherapeutic drugs, such as cisplatin or etoposide . This waIn summary, targeting the IL-6/JAK/STAT3-pathway emerges as a promising way to improve the immune microenvironment in cervical carcinogenesis. However, recent data suggest that these compounds can also interfere with STAT3-induced IRF1 expression in HPV-positive cancer cells, thereby increasing their resistance to chemo- or radiochemotherapy. Therefore, the timing of IL-6/JAK/STAT3-inhibitor administration during therapy appears to be of critical importance. They should not be applied prior to but rather after chemo- or radiochemotherapy of HPV-driven cancers.Over recent years it has become evident that the local microenvironment and particularly the immune system plays a pivotal role in carcinogenesis ,94. In vIn HPV-induced carcinogenesis, there is now ample evidence for a stage-specific interplay between virally-infected keratinocytes and the local immune microenvironment that can determine the course of disease . This kn


Septins constitute a family of GTP-binding proteins, which assemble into non-polar filaments in a nucleotide-dependent manner. These filaments can be recruited to negatively charged membrane surfaces. When associated with membranes septin filaments can act as diffusion barriers, which confine subdomains of distinct biological functions. In addition, they serve scaffolding roles by recruiting cytosolic proteins and other cytoskeletal elements. Septins have been implicated in a large variety of membrane-dependent processes, including cytokinesis, signaling, cell migration, and membrane traffic, and several family members have been implicated in disease. However, surprisingly little is known about the molecular mechanisms underlying their biological functions. This review summarizes evidence in support of regulatory roles of septins during endo-lysosomal sorting, with a particular focus on phosphoinositides, which serve as spatial landmarks guiding septin recruitment to distinct subcellular localizations. Septins constitute a family of small GTPases that assemble into filaments and higher-order structures in a nucleotide-dependent manner  , and might thus act during carrier formation at different organelles. This is supported by several studies that identified septin binding partners with functions during endo-lysosomal sorting Table .2 and PIP3, two PI species found predominantly at the plasma membrane P2-enriched microdomains at the plasma membrane, and to thereby promote the formation of junctions transiently formed with the endoplasmic reticulum during store-operated calcium entry P2 initiates many plasma membrane-derived processes, including endocytosis  in human non-phagocytic cells, and have been proven important for their internalization Ps Figure . SimilarInterestingly, entry of some pathogens additionally depends on parts of the clathrin machinery, including clathrin itself, but also the vesicle fission enzyme dynamin, and a number of accessory proteins known to associate with receptor tyrosine kinases during their endocytosis P  to promote endo-lysosomal fusion. Interestingly, a PIKfyve-dependent pool of PIP2 recruits SEPT2 to fusion sites on Rab7-positive macropinosomes P-positive endosomes acquire a PIPShigella this pool of endosomal PI(5)P impedes EGF receptor degradation P on endosomes. In cells infected with Shigella to target them for autophagy P can also regulate the biogenesis of autophagosomes through a non-canonical, Vps34-independent pathway  receptors upon depletion of SEPT9  .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We examined prevalence and predictors of comprehensive smoke-free household rules  among smokers and nonsmokers in Minnesota. Data came from the 2014 Minnesota Adult Tobacco Survey; weighted analyses consisted of descriptive analyses and multivariate logistic regression analyses. Most adult smokers implemented home-only smoke-free rules (43%) while most nonsmokers implemented comprehensive smoke-free rules . Comprehensive smoke-free rules were more common among people with high socioeconomic status (SES), married people, and people who did not live with a smoker; those with a child in the home were more likely to implement smoke-free homes but not smoke-free cars. Public health practitioners should focus on addressing the majority of smokers who do not implement comprehensive smoke-free household rules, such as low-SES populations, and addressing caregivers who do not implement smoke-free car rules. Smoke-free public policies reduce secondhand smoke in public places; however, private spaces, such as the home and car, are common locations for secondhand smoke exposure . VoluntaData came from the 2014 Minnesota Adult Tobacco Survey (MATS). MATS is a statewide, cross-sectional landline and cellular telephone survey of 9,304 Minnesotans aged 18 years or older, which yielded a combined response rate of 71%. MATS was approved by Minnesota Department of Health’s Institutional Review Board. Survey weights accounted for sampling and ensured statewide representativeness. Smoke-free rules were measured with 2 questions: 1) “Which statement best describes rules about smoking inside your home (excluding porches and garages): not allowed anywhere, allowed some places or at some times, or allowed anywhere”; and 2) “In the vehicles (excluding motorcycles) that you or your family who live with you own or lease, is smoking . . . allowed, sometimes allowed in at least one vehicle, or never allowed in any vehicle.” A composite, 4-category variable captured smoke-free rules: comprehensive (smoke-free home and car), home-only , car-only , and no rules . Covariates were age (4 categories), education (4 categories), low-income (yes or no), race/ethnicity (white or other), sex , marital status (married or not married), living with a child aged less than 18 years (yes or no), location (metropolitan county or nonmetropolitan county), and living with a smoker (yes or no). We used a 3-category measure of smoking intensity: light, moderate, and heavy.svyset command in Stata, version 13 (StataCorp LLC). Stata’s default of F ratios and adjusted Wald tests were used to compare continuous variables across smoke-free rules categories; χ2 statistics and design-adjusted F ratios were used to compare binary variables. A multivariate logistic regression was used to assess characteristics associated with comprehensive smoke-free rules for smokers and nonsmokers separately.Descriptive analyses were used to compare demographic characteristics and smoking behaviors across different household smoke-free rules for smokers and nonsmokers separately. All analyses were conducted using the Most adult smokers implemented home-only smoke-free rules (43%) or did not implement any smoke-free rules (31%) . A smallMultivariate logistic regressions showed that income, marital status, and smoking intensity were related to comprehensive smoke-free rules among smokers. The odds of implementing comprehensive rules decreased for moderate and heavy smokers compared with light smokers . The odds of implementing comprehensive rules increased for married smokers compared with unmarried smokers . Low-income status was negatively associated with implementing comprehensive rules . Having a child in the home aged less than 18 years had a nonsignificant association with comprehensive rules for smokers .For nonsmokers, men were less likely than women to implement comprehensive smoke-free rules . Higher education levels were positively related to comprehensive rule implementation , and low-income status was negatively related to comprehensive rule implementation . Married nonsmokers were more likely to implement comprehensive rules than were unmarried nonsmokers , and the odds of implementing comprehensive rules were lower for nonsmokers who lived with a smoker than with those who did not . Having a child in the home under 18 years had a nonsignificant association with implementing comprehensive smoke-free rules for nonsmokers.This study adds to the literature on voluntary, smoke-free rules by establishing the prevalence and predictors of comprehensive smoke-free rules . We found marked disparities in comprehensive smoke-free rule implementation across smoking status and key demographics.Although 85% of nonsmokers implemented comprehensive rules, only 19% of smokers implemented such comprehensive rules, and 43% implemented home-only rules. Previous research has documented an increase in smoke-free rules in homes , indicatPeople with low income and low education levels were less likely to implement comprehensive smoke-free rules. Practitioners should focus on reducing SES-related barriers to implementing comprehensive smoke-free rules . Tailored health education efforts regarding comprehensive smoke-free rules may help to address these disparities. Living with a smoker was also a barrier to implementation of smoke-free rules (among smokers and nonsmokers); consequently, programs should communicate the importance of comprehensive smoke-free rules by addressing the entire household.Living with a child under age 18 years predicted smoke-free home rules, supporting previous research ,10, but


The aim of this study was to investigate the effect and mechanism by which progesterone regulates uterine contraction in late pregnant ratsProgesterone caused concentration-dependent relaxation of uterine strips that was enhanced compared with control nontreated uterine strips. Uterine strips incubated with progesterone showed a significant increase in TREK-1 mRNA expression and protein level. TREK-1 inhibitor L-methionine partly reversed uterine relaxation caused by the progesterone, while TREK-1 activator arachidonic acid did not cause significant change in progesterone-induced relaxation.Progesterone inhibits uterine contraction and induces uterine relaxation in late pregnancy. The progesterone-induced inhibition of uterine contraction appears to partly involve increased potassium channel TREK-1 expression/activity.Uterus from late-pregnant rats  was isolated, and uterine strips were prepared for isometric contraction measurement. Oxytocin-induced contraction was compared in uterine strips pretreated with different concentration of progesterone. TREK-1 potassium channel inhibitor L-methionine and TREK-1 agonist arachidonic acid were used to determine whether the changes caused by progesterone involve changes in TREK-1 activity. The mRNA and protein expression of TREK-1 in uterine tissues were measured using qPCR and Western blot. During the course of normal pregnancy, the human uterus expands in volume dramatically due to hypertrophy and distension of uterine smooth muscle, thus allowing sufficient space and nutrition for the developing fetus . In contPregnancy is a physiological process encompassing significant hormone-mediated adjustments. One of the most pivotal players during pregnancy is the steroid hormone progesterone . Clinica+) channel, has been described in smooth muscle of human myometrium. TREK-1 decreases myometrial smooth muscle excitability by helping to return depolarized cells to a more negative resting potential and to maintain resting cells near the K+ equilibrium potential . When a statistical difference was observed, the data were further analyzed using Student-Newman-Keuls


Competing endogenous RNA (ceRNA) have received wide attention because they are a novel way to regulate genes through sharing microRNAs (miRNAs) that are crucial for complex processes in many diseases. However, no systematic analysis of ceRNA mechanism in cardiovascular disease (CVD) is known. To gain insights into the global properties of ceRNAs in multi-CVDs, we constructed the global view of mRNA-related ceRNA cross-talk in eight major CVDs from ~2,800 samples. We found common features that could be used to uncover similarities among different CVDs and highlighted a common core ceRNA network across CVDs. Comparative analysis of hub ceRNAs in each network revealed three types of hubs, which might play key roles in diverse biological processes. Importantly, by combining CVD-related pathway genes with ceRNA-ceRNA interactions, common modules that might exert functions in specific mechanisms were identified. In addition, our study investigated a potential mechanistic linkage between pathway cross-talk and ceRNA cross-talk. In summary, this study uncovered and systematically characterized global properties of mRNA-related ceRNA cross-talks across CVDs, which may provide a new layer for exploring biological mechanisms and shed new light on cardiology. MiRNAs are typically 22 nucleotides long and negatively regulate or repress mRNAs or non-coding transcripts by guiding associations between the RNA-induced silencing complex (RISC) and targeting RNAs4. Some studies have shown that a group of mRNA transcripts are involved in a ceRNA cross-talk by competing for common miRNA binding sites 8. Some ceRNA cross-talks have been validated from the first discovery of ceRNA in cancer, such as PTEN9, FOXO110 and AEG-111. An increasing number of studies have attempted to uncover the mechanism of CVDs in the level of ceRNAs13. For example, the circRNA HRCR and its ceRNA partners could inhibit cardiac hypertrophy and heart failure by functioning as miRNA sponges of miR-22314. Wang et al. found that the lncRNA CHRF act as an endogenous sponge of miR-489, down-regulating miR-489 expression and regulating Myd88 expression in cardiac hypertrophy15. The lncRNA APF is a sponge of miR-188-3p that decreases degradation of ATG7, which regulates autophagy and myocardial infarction16. These studies suggest that ceRNA cross-talk is crucial in miRNA-mediated interactions in CVDs and systematic investigation of the ceRNA cross-talk across CVDs is needed.MicroRNAs (miRNA) play crucial regulatory roles in CVDs17. CVD composed more than 10 disease subtypes and investigating the common or specific features across various diseases is important. The rapid development of high-throughput experimental techniques such as microarray and RNA-seq can detect the expression of mRNA transcripts. These techniques have contributed to integrative analysis of molecular ceRNA interactions using gene expression correlations. Studies also provided supports to the research of ceRNA, such as open source data and computational methods. For instance, the open source database starBase provides CLIP-seq supported miRNA-mRNA interactions18. Our previous study investigated lncRNA-mRNA ceRNA cross-talks by integrative analysis of gene dysfunction and CLIP-seq-supported miRNA-mRNA/lncRNA interactions in cardiac hypertrophy19. In addition, a study proposed a new computational method to identify the sponge interaction by integrating gene co-expression information in breast cancer20. Other studies focused on cancer-related ceRNA networks constructed using gene co-expression levels22. A landscape of mRNA-related ceRNA interactions across 20 cancer types was constructed through integrating TCGA gene expression data23. Wang et al. identified lncRNA-associated competing triplets across cancers using TCGA data24. TCGA stored the data only on multiple cancers. But in the field of CVDs, no database systematically stores gene expression data. Thus global research on CVDs is difficult. Luckily, we obtained gene expression data on CVDs from GEO database, which stored numbers of mRNA-related expression profiles. Compared with the TCGA database, data from GEO is dispersive, thus we can only collect data manually. By performing efficient bioinformatics methods, we obtained gene expression data for investigating the ceRNA cross-talks in multiple CVDs.CVD, a leading cause of death, encompasses a broad range of conditions from myocardial infarction to congenital heart disease; most CVDs are heritable In our present study, we performed an integrated analysis of 2,884 samples from eight major CVDs to identify mRNA-related ceRNA cross-talk. First, ceRNA cross-talk networks of each gene expression profile were constructed. We systematically analyzed the topological features and characterized common properties in these ceRNA networks. A common core ceRNA-ceRNA interaction network was identified for various CVDs. After merging the disease associated ceRNA networks, we mapped the CVD-related pathway genes into the networks to identify the common modules that might be crucial for the processes of CVDs using in depth analysis of network structures. In addition, we also found that the ceRNA cross-talks might drive the cross-talks of pathways.To systematically evaluate the potential role of miRNA-mediated ceRNA cross-talk in multiple CVDs, we performed a two-stage analysis to construct ceRNA cross-talk landscapes for each CVD-associated gene expression profile Fig. . We merg26. Our analysis of the interactions of ceRNA nodes showed that ceRNAs with higher degree were more highly co-expressed than others in most ceRNA networks . As a result, we identified 37,332 ceRNA cross-talk pairs that were associated with pathways. We then split these ceRNA pairs into two groups for a total of 2,366 CPWPs and 34,966 CPBPs. Most ceRNA cross-talks were involved in CPBP, indicating that ceRNA might play an important role in pathway cross-talk, which was regarded as an important mechanism in disease development processes. Interestingly, some ceRNA belonged to most of pathways, such as the MAPK family, PIK3CA and TNF, which mediate important pathway cross-talks and are demonstrated to be known disease genes. Next, we investigated the properties of ceRNAs involved in these pathway-associated cross-talks and other genome genes. As a result, pathway-associated ceRNA transcripts were longer and had more exons than other transcripts , CHD, DCM, HCM, HF and PH. We re-analyzed all these disease related ceRNA networks, including degree analysis and hub distributions. Results showed a high similarity compared with results of 8 disease related ceRNA networks. For example, we performed degree cumulation distributions for each disease network . GEO is a public functional genomics data repository supporting MIAME-compliant data submissions. In total, our study investigated eight major CVDs: coronary artery disease(CAD), hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), ischemic cardiomyopathy (ICM), heart failure (HF), myocardial infarction (MI), pulmonary hypertension (PAH) and congenital heart disease (CHD) in 21 gene expression datasets  immunoprecipitation coupled with high-throughput sequencing (CLIP-Seq) could identify the genome-wide interaction of miRNAs and their targeting RNAs41. starBase V2.0 database is designed for decoding interaction network via integrating large-scale CLIP-Seq  data. MiRNA targets of starBase V2.0 were predicted by five target predicted algorithms, including TargetScan, miRanda, Pictar, PITA, and RNA22.In this study, CLIP-seq-supported miRNA-mRNA interactions were downloaded from starBase V2.0 databasehttp://www.kegg.jp/kegg/) is a collection of manually drawn pathway maps representing our knowledge on the molecular interaction and reaction networks. The sub-catalog “cardiovascular diseases” collected by KEGG pathway database was used in this study. The sub-catalog had four pathways drawn in the in KEGG database directly; we selected the related pathways that occurred in each of the four pathway maps as the CVD-related pathways indirectly. In total, we obtained 16 CVD-related pathways. Genes annotated in the 16 pathways were extracted by the R package of “iSubpathwayMiner”42, which was developed from our research group.KEGG PATHWAY  CVD-specific hub nodes; 2) differential hub nodes; and 3) common ceRNA hub nodes. Specially, the hub nodes that only occurred in one ceRNA network were defined as CVD-specific hub nodes. Differential hub nodes were the hub nodes occurred in more than one ceRNA network but their neighbors changed between different diseases. And common ceRNA hub nodes, that was defined as the ceRNAs were hub nodes in more than one ceRNA with similar neighbors. For the first category, the ceRNAs only ranked in top 10% of highest degrees in one ceRNA networks, for the other two categories, we calculated the similarity of their interacting neighbors between pairs of ceRNA networks by the method of Simpson index. If a hub node with Simpson index > 0.8 for at least one pair of ceRNA networks, we assigned the hub node into the third category. Otherwise, we grouped the hub nodes into the second category.Some studies have demonstrated that hub nodes with higher degrees in biological networks were more important than others. We selected the top 10% of nodes with the highest degrees as the hub nodes in this study. To analyze the distribution of hub nodes across all CVDs, we classified the hub nodes into three categories according to the strategy proposed by Xu First, we merged all 21 ceRNA networks of gene expression profiles into eight ceRNA networks of diseases according to the disease name. We obtained the genes from CVD-related KEGG pathways using “iSubpathwayMiner” and mapped all the genes into the ceRNA networks of disease. Edges mediated by two KEGG pathway genes were extracted. Finally, CVD pathway-associated ceRNA networks were constructed.48. We reserved the scale of community that was more than 3 and less than 300.For each ceRNA network of gene expression profiles, we identified the modules by applying the method called multi-level optimization of modularity in R package “igraph”. This method was based on the modularity measure and a hierarchical approach. Modules that identified from the method were defined as communitieshttp://www.cfinder.org/), a fast program for locating and visualizing overlapping, to find common modules defined as communities. Similarly, CVD-specific ceRNA modules were identified from the sub-network that composed of CVD-specific hub nodes for each CVD pathway-associated ceRNA network.To identify modules of common hub ceRNAs in the CVD pathway-associated ceRNA networks, all the CVD pathway-associated ceRNA networks were merged and we extracted the sub-network edges linked by common hub nodes. We performed CFinder , defined as one ceRNA pair only occurred in one pathway. Briefly, one ceRNA pair: gene1-gene2, both gene1 and gene2 occurred in pathway A. And the other one was that ceRNA pairs between-pathways (CPBP), which was defined as two ceRNAs that in one ceRNA cross-talk not occurred in same pathway. For example, one ceRNA pair: gene1-gene2, gene1 belongs to pathway A, gene2 not belongs to pathway A.Supplementary materialsSupplementary Table S6


Prostate cancer (PCa) is a leading cause of male cancer-related deaths. A significant fraction of prostate tumors are very aggressive, often metastasizing to bone, causing significant morbidity and mortality. Also, PCa is associated with high rates of recurrence, often attributed to the existence of cancer stem cells. Epithelial-mesenchymal transition (EMT), a process characterized by decreased expression of epithelial genes and increased expression of mesenchymal genes, plays a critical role in tumor invasion, metastasis and recurrence. In PCa, EMT has been implicated particularly in the context of metastatic disease and microRNAs have emerged as critical post-transcriptional regulators of PCa EMT. In this review, we summarize the role of miRNAs in PCa EMT that play a role in progression, metastasis and recurrence. Studies till date suggest that microRNAs mediate efficient and reversible control of PCa EMT via multiple mechanisms including either by (i) directly repressing single or multiple EMT-TFs or regulating cytoskeletal components  or (ii) regulating key signaling pathways involved in EMT. Oncogenic microRNAs often act as EMT promoters by repressing epithelial characteristics and tumor suppressive miRNAs act by inhibiting mesenchymal progression. Further, EMT is mechanistically linked to stem cell signatures in PCa and several miRNAs implicated in EMT have been reported to influence PCa stem cells. Loss of EMT-inhibiting miRNAs and/or gain of EMT promoting miRNAs lead to induction of PCa EMT, leading to tumor progression, metastasis and recurrence. Restoring expression of tumor suppressive miRNAs and inhibiting oncogenic miRNAs represent potential therapeutic opportunities to prevent disease metastasis and recurrence. Prostate cancer (PCa) is the most commonly diagnosed cancer among men , 2. It iEpithelial cancer cells convert to motile, mesenchymal cells by undergoing an epithelial-mesenchymal transition (EMT) –14 that Several EMT-transcription factors (EMT-TFs) repress the expression of E-cad and other epithelial markers and induce the expression of mesenchymal markers . There aMicroRNAs recently emerged as important regulators of EMT and MET in various cancers , 18. Micin vivo  as  as 18] aThe miR-34 family of tumor suppressive miRNAs include miR-34a/b/c that are encoded by two different genes. miR-34a precursor is transcribed from chromosome 1 while miR-34b and miR-34c precursors are co-transcribed from a region on chromosome 11 . miR-34 miR-23b is a methylation-silenced tumor suppressor in prostate cancer that works by directly targeting proto oncogene Src kinase and Akt  is a frequently overexpressed oncogene that is activated by fusion events between promoter sequences of the TMPRSS2 and coding sequences of ERG. Kao f miR-30 . miR-30 f miR-30 . Furtherf miR-30 .miR-100 negatively regulated EMT in PC3 and DU145 cells and its expression was negatively correlated with bone metastasis of prostate cancer patients . DownregmiR-29b is downregulated in PCa cells as compared to normal prostate epithelial cells, and its expression was further lowered in metastatic prostate cancer cells . Upon mimiR-195 is downregulated in prostate cancer tissues and low miR-195 is significantly associated with high Gleason score, biochemical recurrence and metastasis , 98. PromiR-223 is significantly downregulated in PCa tissues and restoration of miR-223 led to inhibited cell proliferation/invasion . miR-223in vitro and lung metastasis in vivo by directly targeting the fibroblast growth factor receptor 1 (FGFR1) gene [miR-573 is downregulated in metastatic PCa in comparison to primary PCa . miR-573R1) gene . FGFR1 pR1) gene , 103. FuR1) gene .In prostate cancer, various oncogenic microRNAs often repress epithelial characteristics or promote EMT as summarized below.Oncogenic miRNAs can promote EMT by augmenting the expression of EMT-TFs indirectly as exemplified by miR-301a or by directly targeting cytoskeletal components .In a recent study, Nam et al. showed that miR-301a induces EMT in prostate cancer cells by directly targeting and inhibiting the tumor suppressor p63. The loss of p63 results in the downregulation of tumor suppressive miR-205, causing upregulation of ZEB1 and ZEB2  cell lines PC3 and Du145 . TreatmePC cells .In addition, oncogenic miRNAs may repress tumor suppressor genes or act by repressing inhibitors of signaling pathways involved in EMT as summarized below Table .miR-21 is commonly upregulated in various cancers, including prostate cancer . miR-21 et al showed that miR-32 also targets BTG2 and that their expression levels are inversely correlated in castration-resistant prostate cancer (CRPC) [Jalava r (CRPC) . miR-32 r (CRPC) .miR-154* and miR-379 are members of the delta-like 1 homolog-deiodinase, iodothyroxine (DLK1-DIO3) cluster - a cluster that plays an important role in EMT and embryonic development. Gururajan et al. demonstrated that both miR-154* and miR-379 have elevated expression in bone metastatic PCa cell lines and tissues and miR-379 expression correlated with progression-free survival of PCa . OverexpmiR-331-3p expression promotes EMT as its overexpression in the PC3 cell line led to upregulation of mesenchymal markers such as vimentin and N-cadherin, and downregulation of epithelial markers such as E-cadherin and desmoplakin . FurthermiR-331 expression is elevated in PCa cases with high Gleason score and was shown to be controlled by Syndecan-1 .miR-129-3p has been reported to be upregulated in metastatic PCa cells . miR-129miR-409-3p/-5p expression is elevated in bone metastatic PCa cell lines and in PCa tissues with higher Gleason scores . High miProstate cancer recurrence and progression has been associated with the existence of prostate cancer stem cells (CSC) or tumor-initiating cells (TIC). These cells are refractory to current therapies  and are In conclusion, the role of miRNAs in PCa EMT is emerging and warrants further investigation. Studies till date suggest that microRNAs mediate efficient and reversible post-transcriptional control of prostate cancer EMT via multiple mechanisms including direct targeting of EMT-TFs, affecting signaling pathway components controlling EMT or by directly modulating cytoskeletal components. Oncogenic microRNAs often act as EMT promoters by repressing epithelial characteristics and tumor suppressive miRNAs act by inhibiting mesenchymal progression. Loss of EMT-inhibiting miRNAs and/or gain of EMT promoting miRNAs lead to induction of PCa EMT, leading to tumor progression, metastasis and recurrence. Restoring expression of tumor suppressive miRNAs and inhibiting oncogenic miRNAs represent therapeutic opportunities to prevent PCa metastasis and recurrence.


Dear Editor,1 EMT allows polarized, immotile epithelial cells to transform into motile mesenchymal cells.2 This switch is mediated by regulatory factors such as transcription factor (TF), microRNA (miRNA) and long non-coding RNA (lncRNA) at transcriptional and post-transcriptional levels.4 These regulatory factors play co-regulational roles on the EMT process and the regulatory loops act as motifs, which are the basic units in the regulatory network.5 However, a database with comprehensive EMT regulatory relationships is still lacking. EMT-Regulome comprehensively collected the interactions between regulators and targets, including TF-target, miRNA-target, miRNA-lncRNA, TF-lncRNA and TF-miRNA and identified 10 types of co-regulation motifs for EMT process  is a fundamental cellular process that governs biological processes, such as embryonic development, tumor cell metastasis, organ fibrosis and tissue regeneration. process . EMT-Reg6 which collected experimentally verified EMT-related genes from literatures, were defined as EMT-related motifs in the EMT-Regulome database. The statistics of motifs are shown in http://js.cytoscape.org).In current version, EMT-Regulome contains 1740 miRNAs, 8783 lncRNAs and 13 025 coding genes, which includes 370 TF coding genes and 350 EMT-related genes . We collet al.7 have classified the TCGA ovarian cancer samples into integrated epithelial (iE) subtype and integrated mesenchymal (iM) subtype labels. Here, the activated motifs were defined as the nodes in motifs that were differentially expressed in iM compared with the iE ovarian cancer samples (T-test) and the edges in motifs were differentially co-expressed in iM type (Pearson correlation test). All the activated EMT-related motifs in ovarian cancer are available in the download page or by searching specific nodes with statistical control in search page.As a case analysis, the EMT-Regulome database was applied to identify potential activated EMT motifs in the mesenchymal subtype of ovarian cancer by employing expression profiles from The Cancer Genome Atlas (TCGA). Yang In conclusion, EMT-Regulome provides a comprehensive landscape of EMT-related regulation motifs to facilitate the investigation of the mechanism of EMT involved in the pathological and physiological processes. The features of EMT-Regulome include, (i) comprehensively integrated different EMT-related regulatory relationships from multiple databases; (ii) systematically provided EMT-related motifs, such as feed-forward loop and ceRNA with statistical control; (iii) all the EMT-related regulatory relationships can be visualized as network and be downloaded.


Poly(ADP-ribose) polymerases are a family of DNA-dependent nuclear enzymes catalyzing the transfer of ADP-ribose moieties from cellular nicotinamide-adenine-dinucleotide to a variety of target proteins. Although they have been considered as resident nuclear elements of the DNA repair machinery, recent works revealed a more intricate physiologic role of poly(ADP-ribose) polymerases with numerous extranuclear activities. Indeed, poly(ADP-ribose) polymerases participate in fundamental cellular processes like chromatin remodelling, transcription or regulation of the cell-cycle. These new insight into the physiologic roles of poly(ADP-ribose) polymerases widens the range of human pathologies in which pharmacologic inhibition of these enzymes might have a therapeutic potential. Here, we overview our current knowledge on extranuclear functions of poly(ADP-ribose) polymerases with a particular focus on the mitochondrial ones and discuss potential fields of future clinical applications. Basal metabolic activity as well as environmental factors lead to more than 20,000 DNA alterations per cell every day . Since sAlthough PARP inhibitors, like Olaparib or Veliparib, failed to deliver expected results in clinical trials, they led to the discovery of a number of PARP-mediated extranuclear effects including mitochondrial functions or crosstalk with canonical signaling pathways –4. Here,PARPs are expressed ..2+-stimu2+-mediated in primary culture of newborn cardiomyocytes and that PARP-1 activation follows Ca2+ release from perinuclear stores in depolarized primary culture of rat brain cortical neurons suggest that, at least in certain cellular contexts, Ca2+ may be one of the candidate mediators of PARP-1-activating stimuli [2+ levels, possibly by utilizing or mimicking the effects of the AKT/PKB pathway thereby contributing to mitochondrial integrity and cell survival.Although detailed mechanisms are yet to be determined, observations that the angiotensin II-induced PARP-1 activation is Ca stimuli , 114. Th2+-induced PTP opening [Mitochondrial function and morphology are intimately linked. Indeed, over-expression of the mitochondrial fission proteins DRP1 or hFIS1 increases susceptibility to Ca opening . Convers opening . Intrigu opening , 118. OP opening , 120. OP opening , 122. Th opening , 124. Mo opening .The complex effects of the AKT/PKB pathway on mitochondria is further illustrated by recent findings that AKT/PKB induces trafficking of energetically active mitochondria to the cortical cytoskeleton of tumor cells leading to lamellipodia formation, supports turnover of fatty acid complexes and random cell migration . IntriguL, attenuating their pro-survival activity that facilitates cytochrome c release and collapse of the mitochondrial membrane potential [The AKT/PKB pathway, however, is not the only signaling mechanism associated with the regulation of mitochondrial morphology and PARP-1. Activation of JNK and p38 MAP kinases has also been reported in response to PARP-1 and both kinases are involved in the regulation of mitochondrial dynamics and function , activation of AKT/PKB and disruption of NF-κB-mediated inflammation adding new aspects to the therapeutic use of these compounds in chronic inflammatory conditions like vascular disease or mellitus , 169.The unexpectedly tangled cellular effects of PARP clearly call for further investigations. These efforts not only hold out the promise of enhancement of the therapeutic concept of PARP inhibition but a better understanding of the complex PARP-1 interactome as well.


Plants respond to pathogens using an innate immune system that is broadly divided into PTI (pathogen-associated molecular pattern- or PAMP-triggered immunity) and ETI (effector-triggered immunity). PTI is activated upon perception of PAMPs, conserved motifs derived from pathogens, by surface membrane-anchored pattern recognition receptors (PRRs). To overcome this first line of defense, pathogens release into plant cells effectors that inhibit PTI and activate effector-triggered susceptibility (ETS). Counteracting this virulence strategy, plant cells synthesize intracellular resistance (R) proteins, which specifically recognize pathogen effectors or avirulence (Avr) factors and activate ETI. These coevolving pathogen virulence strategies and plant resistance mechanisms illustrate evolutionary arms race between pathogen and host, which is integrated into the zigzag model of plant innate immunity. Although antiviral immune concepts have been initially excluded from the zigzag model, recent studies have provided several lines of evidence substantiating the notion that plants deploy the innate immune system to fight viruses in a manner similar to that used for non-viral pathogens. First, most R proteins against viruses so far characterized share structural similarity with antibacterial and antifungal R gene products and elicit typical ETI-based immune responses. Second, virus-derived PAMPs may activate PTI-like responses through immune co-receptors of plant PTI. Finally, and even more compelling, a viral Avr factor that triggers ETI in resistant genotypes has recently been shown to act as a suppressor of PTI, integrating plant viruses into the co-evolutionary model of host-pathogen interactions, the zigzag model. In this review, we summarize these important progresses, focusing on the potential significance of antiviral immune receptors and co-receptors in plant antiviral innate immunity. In light of the innate immune system, we also discuss a newly uncovered layer of antiviral defense that is specific to plant DNA viruses and relies on transmembrane receptor-mediated translational suppression for defense. Figure 1). PRRs recognize conserved structural motifs present in microbes, which are known as microbe- or pathogen-associated molecular patterns (MAMPs/PAMPs), or endogenous danger signals released by the plant during wounding or pathogenic attack, which are termed damage-associated molecular patterns . These intracellular receptors directly or indirectly recognize effectors secreted by pathogens into the host intracellular environment and activate effector-triggered immunity . Activation of NIK1-mediated antiviral signaling leads to translocation of the ribosomal protein L10 (RPL10) to the nucleus, where it interacts with L10-INTERACTING MYB DOMAIN-CONTAINING PROTEIN (LIMYB) to fully repress expression of translational machinery-related genes and global host translation. Begomovirus mRNAs are unable to escape this translational regulatory mechanism of plant cells and hence are not efficiently translated, which compromises infection upon activation of NIK1-mediated defense. Although the NIK1-mediated defense response is remarkably dissimilar from the PTI response, structural components and activation of the NIK1 immune receptor as well as its interaction with virus infection exhibit features reminiscent of the plant innate immunity mechanism.An additional recently uncovered virus-specific defense mechanism relies on suppression of host translation mediated by the transmembrane immune receptor NUCLEAR SHUTTLE PROTEIN-INTERACTING KINASE 1 (NIK1), which was first identified as a virulence target of begomovirus NSP , Tomato bushy stunt virus (TBSV), Cauliflower mosaic virus (CaMV) and Tomato mosaic virus  product, can lead to the hypersensitive reaction (HR). HR is considered a resistance response against several different pathogens that, to the some extent, occurs through similar mechanisms. Similar to non-viral infections, the HR response during viral infection is initiated by direct or indirect Avr-R interactions and is frequently associated with accumulation of SA in both infected and non-infected tissues . HR is auring HR . Althougus ToMV; , 2009. Fus ToMV; , 2009.Table 1). Based on their variable N-terminal domain, these plant NB-LRR proteins are further classified into coiled-coil (CC)-NB-LRR or Toll/interleukin 1 receptor-like (TIR)-NB-LRR protein families ] proteins that mediate resistance via specific (direct or indirect) recognition of a virus Avr factor  (Table 1families . Most offamilies . Recognifamilies  or throufamilies . In the families . Alternafamilies . TherefoTable 1)   . AnotherTable 1) , PHYTOALTable 1) , 2005 anTable 1) . The HRTTable 1) . DisruptTable 1)  and EDS1Table 1)  protein N gene (for necrotic-type response), which confers resistance against TMV and encodes a TIR-NB-LRR protein, was the first identified R gene . One such example is the tomato Tm-1 gene, which confers dominant resistance to ToMV and contains two conserved domains: an uncharacterized N-terminal region (residues M1–K431) and a TIM-barrel-like C-terminal domain . The Ty-1 and Ty-3 genes are allelic and code for an RDR of the RDRc type, which has an atypical DFDGD motif in the catalytic domain (Ty-1/Ty-3 plants display enhanced siRNA levels that coincide with hypermethylation of the TYLCV V1 (CP) promoter, indicating that Ty-1-based resistance against TYLCV involves enhanced transcriptional gene silencing.A few dominant resistance genes encoding the non-NB-LRR class of proteins have been characterized; these proteins have been found to function as sensors of virus infection but do not induce typical ETI-like defense responses, such as HR (c domain . The mecc domain . AccordiTable 1), which specifically recognize viral effectors or Avr factors and utilize signaling modules such as SGT1/RAR1/HSP90 and EDS1/PAD4/SAG101 complexes to mediate resistance responses, similar to non-viral pathogens. Therefore, plants appear to have evolved strategies and signaling modules to defend themselves against a large spectrum of pathogen types, such as bacteria, viruses and fungi. This interpretation allows us to integrate some aspects of the antiviral immune concepts into the typical bacterial and fungal immunity models to classify viral effectors and viral ETI.In summary, most antiviral dominant resistance genes so far characterized encode typical NB-LRR R proteins , have important roles in antiviral defense via recognition of a different range of MAMPs, such viral RNA and DNA . In contnfection . Consistt manner . The Ararus PPV; . Furtherus BPMV; , and chius BPMV; .Oilseed rape mosaic virus (ORMV) showed significantly reduced viral accumulation in treated leaves. Interestingly, dsRNA-mediated PTI appears to be independent of the RNA silencing pathway but does involve the co-receptor kinase SERK1. These findings relate membrane-associated signaling events with dsRNA-mediated PTI in plants  and virus-derived dsRNA have been shown to function as viral PAMPs in Arabidopsis and to induce the PTI pathway . Indeed,n plants . Althougin planta during infection (Table 1). These observations suggest that plant viruses also fit into the zigzag model of co-evolving pathogenic virulence strategies and plant defense responses that shape the two-branched innate immune system .The transmembrane receptor NIK was first identified as a virulence target of Nuclear Shuttle Protein (NSP) from e family . Similare family . NeverthNUCLEAR SHUTTLE PROTEIN-INTERACTING KINASE 1-mediated antiviral signaling is activated upon perception of begomovirus infection, which leads to phosphorylation of the NIK1 kinase at key threonine residues at positions 468 and 474 . Thr-468In general, ligand-dependent phosphorylation and activation of RLKs require homo or heterodimerization of the receptors. In the case of BAK1 and SERK1, compelling evidence has revealed that they function primarily as co-receptors for receptor signaling not only in defense but also in development . As a mein vitro and in vivo with the kinase domain of NIK1 to suppress NIK1 activity (Bean dwarf mosaic virus (BDMV) has been demonstrated to function as an Avr gene and elicit HR in Phaseolus vulgaris  infection . Activated NIK1 mediates phosphorylation and consequent translocation of RPL10 to the nucleus, where it interacts with LIMYB to fully repress RP gene expression. Prolonged down-regulation of RP gene expression leads to suppression of global host translation. DNA viruses, such as begomoviruses, cannot escape this translational regulatory mechanism of plant cells, and viral mRNAs are not translated efficiently, thereby compromising infection. NSP acts as a virulence factor and suppresses the kinase activity of NIK1 to overcome the NIK1-mediated immune response. NSP from the begomovirus BDMV has also been shown to function as an Avr factor that activates typical ETI responses in resistant bean genotypes. Therefore, NSP may link the suppression of NIK1 signaling with activation of ETI responses in accordance with the zigzag evolutionary model of plant innate immunity, although the NIK1-mediated antiviral signaling may represent a new evolved branch of plant antiviral immunity, which relies on suppression of translation for defense.Although NIK1 is structurally similar to SERKs, the mechanism of NIK1-mediated antiviral defense is distinct from that of BAK1-mediated PTI. The current model for NIK1-mediated antiviral signaling states that, in response to virus infection, NIK1 undergoes homo- or heterodimerization to promote phosphorylation of the activation loop  and NB-LRR host targets (R proteins) and their mode of action in activating ETI and/or suppressing PTI will help to define the evolutionary pressure acting upon the host and viruses and to determine how to deploy the immune system for more efficient control of virus infection. We also need to define NIK1-mediated suppression of translation as a general or virus-specific antiviral strategy in plants. To date, a sustained NIK1 pathway has been shown to be effective against begomoviruses, one of the largest groups of plant DNA viruses, which cannot circumvent the regulatory mechanism of host translation. In this regard, the intrinsic capacity of agronomically relevant crops to withstand the deleterious effects of suppression of global translation must be considered as a relevant agronomic trait if we are to use the translational control branch of NIK1-mediated antiviral signaling for crop protection against begomoviruses.BG and IC wrote the first draft of the manuscript; JM wrote the ETI section; AS edited the manuscript and EF edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A new method for converting commodity polymeric materials into value-added specialty materials was developed via site-selective and chemoselective transesterification of block copolymers, statistical copolymers, and homopolymers. The direct transformation of commercially available commodity polyacrylates into value-added materials was achieved. We demonstrate how 1,5,7-triazabicyclo[4.4.0]dec-5-ene, serving as a nucleophilic catalyst, can be used to catalyze acyl substitution reactions of acrylic polymers in the presence of alcohol and amine nucleophiles. Furthermore, we found that organocatalytic transesterification exhibits high selectivity towards sterically unhindered esters, thus providing a new route towards site-selective acyl substitution of macromolecular materials. Combining this methodology with reversible-deactivation radical polymerization (RDRP) techniques such as reversible addition–fragmentation chain-transfer (RAFT) polymerization allowed for the precise functionalization of sterically-differentiated acrylic copolymers and polymeric chain ends. We envision this approach to expedite functional polymer synthesis and provide access to functional macromolecules prepared from inexpensive, hydrolytically-stable polymeric precursors. Furthermore, chain-end selective transesterification of PMMA was achieved with the absence of competing side-chain transesterification, highlighting the high selectivity of TBD-catalyzed transesterification.via organocatalysis. The approach we report provides a new straightforward method of chemoselectively and efficiently modifying vinyl polymers derived from acrylates and methacrylates. We envision this chemistry, which relies on a platform suitable to large-scale synthetic modification, will streamline access to functional materials derived from polyacrylates.We reasoned that functional polymers could be directly synthesized from hydrolytically-stable, commercially-available, commodity polymers Mn GPC MALS = 9800 g mol–1, Đ = 1.2)  polymerization) Fig. S1.When PMA was subjected to 30 mol% TBD in the presence of benzyl alcohol  at 80 °C in a closed reaction flask, incomplete conversion was observed, likely due to competitive transesterification of benzyl alcohol and the methanol produced over the course of the reaction.P1), as confirmed by 1H nuclear magnetic resonance (NMR) spectroscopy by the appearance of benzylic methylene protons exhibiting a chemical shift (δ) at 5 ppm  was carried out using diethylene glycol methyl ether. We then studied the compatibility of TBD-catalyzed transesterification in the presence of tert-butyloxycarbonyl (boc) protecting groups. Transesterification reactions with N-boc-ethanolamine (P5) led to boc deprotection and subsequent gelation under reflux conditions in toluene; however, when the reaction was conducted at 80 °C, conversion of ca. 73% was observed, suggesting this approach is compatible with urethane functional groups and protected amines. 9-Anthracenemethanol however showed low incorporation (ca. 33%) into PMA, even at a higher stoichiometric ratios. This reduced efficiency is likely attributed to steric effects of the anthracenyl groups preventing subsequent nucleophilic attack of neighboring methyl esters once appended to the polymer chain.We next sought to introduce functional alcohols capable of downstream functionalization. Both resultant polymers tert-butanol as model nucleophiles. Cyclohexanol appeared to undergo efficient functionalization (ca. 81%), although giving lower conversion due to sterics. In the case of tert-butanol, no transesterification product was observed. Lastly, we wanted to study phenol as a nucleophile capable of acyl substitution. We anticipated hydroxyl nucleophiles with low pKa values would exhibit efficient transesterification kinetics due to the general base activation mechanism between TBD and the approaching nucleophile.Next, we further studied the effect of sterics on this methodology by choosing sterically hindered alcohols such as cyclohexanol and n-butyl acrylate) , poly(tert-butyl acrylate) , and poly(methyl methacrylate) . Benzyl alcohol and benzylamine were chosen as model nucleophiles to probe the reactivity , PnBMA-co-PMA (CP2), PPEGMA-co-PMA (CP3), and PtBA-co-PMA (CP4).Copolymers of methyl acrylate with various methacrylic comonomers were synthesized using free radical polymerization to yield PMMA-co-PMA (tBA-co-PMA (n-butyl methacrylate and poly(ethylene glycol) monomethyl ether methacrylate, lower conversion was observed under similar reaction conditions  transesterification in the presence of sterically hindered comonomers. For PMMA-co-PMA  and PtBAA-co-PMA , only thb-PMA, was synthesized using RAFT polymerization . We then sought to generate an amphiphilic block copolymer product through the addition of diethylene glycol methyl ether (DEG ME) as a hydrophilic alcohol in the presence of TBD. Transesterification of the PMA block with DEG ME achieved high conversion  transesterification allowed for quantitative functionalization and proved to be a suitable strategy for the efficient installation of functional alcohols. As compared to approaches that involve functional precursors that contain activated esters, the strategy reported here relies on polyacrylates that effectively have extended shelf-lives due to their hydrolytic stability. Furthermore, this method provided access to site-selective polymer functionalization through chemoselective transesterification of block copolymers, statistical copolymers, as well as homopolymers.A variety of synthetic applications can be envisioned utilizing this methodology such as the chemical upcycling of adhesives and coatings waste into value added materials. Such methodology developments are of growing importance due to issues concerning the long-term limitations of materials derived from nonrenewable resources. In addition to chemical repurposing of waste materials, this strategy can be used as a new synthetic tool in the chemoselective activation of polymeric materials to precisely functionalize well-defined macromolecules.There are no conflicts to declare.Supplementary informationClick here for additional data file.


Gallium-67 scintigraphy is useful for detecting active inflammation. We show a 66-year-old female patient with atrial fibrillation and diffuse thickening of the left atrial wall due to acute myocarditis, who presented diffuse abnormal accumulation of gallium-67 in the left atrium on single photon emission computed tomography/computed tomography (SPECT/CT) fusion images. In the second gallium-67 scan 2 months after the first scintigraphy, the abnormal accumulation in the heart was no longer visible. Gallium-67 SPECT/CT images helped understanding the disease condition that temporary inflammation in the left atrium caused atrial fibrillation. Gallium-67 citrate injected intravenously binds to transferrin, and it is incorporated into the transferrin receptor of inflammatory cells or malignant tumor cells. Because of the above-mentioned properties, gallium-67 scintigraphy is used to detect a focus of active inflammation or malignant lesion. In Japan, F18-fluorodeoxyglucose positron emission tomography (FDG-PET) examination is now not reimbursed by Japanese health insurance system for the diagnosis of active inflammation except for cardiac sarcoidosis, but, globally, FDG-PET is more frequently used for the diagnosis of active inflammation and malignant disease because of its superior spatial resolution. Regarding heart disease, however, prolonged fasting should be required before FDG-PET to reduce physiological FDG accumulation in the heart. Gallium-67 scintigraphy is useful for examination of heart disease including cardiac sarcoidosis and acute myocarditis because gallium-67 does not accumulate physiologically in the heart . Herein,A 66-year-old woman visited our hospital with a chief complaint of palpitations. Since electrocardiogram showed paroxysmal atrial fibrillation and echocardiography showed a thrombus in the left atrium, she was admitted to receive treatment. Echocardiography also showed pericardial effusion and circumferential thickening of the left atrial wall. The thrombus in the left atrium revealed high echoic mass, while circumferential thickening of the left atrial wall revealed low echoic lesion in echocardiography. Thus, these two parts were completely different components. To examine active inflammation or malignancy for the thickened left atrial wall, gallium-67 scintigraphy was performed. The frontal planar image showed abnormal accumulation of radioisotope (RI) in the chest . The sinβ-blocker for atrial fibrillation achieved favorable heart rate control. In the second gallium-67 scan 2 months after the first scintigraphy, the abnormal accumulation in the heart was no longer visible on the planar and SPECT/CT images (Anticoagulant therapy for the thrombus in the left atrium resulted in its dissolution, and administration of a T images . EchocarGallium-67 scintigraphy is useful for detecting active inflammation or malignant lesion. Regarding cardiac disease, it plays an important role in examining inflammatory disease including acute myocarditis, cardiac sarcoidosis, and cardiac amyloidosis –3.Several studies report the gallium-67 scan to be useful in the diagnosis of acute myocarditis , 4. The Some reports examined accumulation of RI in cardiac sarcoidosis lesions using gallium-67 SPECT/CT and FDG-PET/CT , but theThis patient presented with atrial fibrillation on the electrocardiogram, left atrial thrombosis, and diffuse thickening of the left atrial wall on the echocardiography. SPECT/CT fusion images of gallium-67 scan showed corresponding diffuse abnormal accumulation of RI in the left atrial wall. These findings were useful for the localization of inflammation due to acute myocarditis. When abnormal accumulation of gallium-67 is observed in the heart on planar images, an additional SPECT/CT scan may be useful for evaluation of accurate localization.


Sarcoma tumors are rare and heterogeneous, yet they possess many characteristics that may facilitate immunotherapeutic responses. Both active strategies including vaccines and passive strategies involving cellular adoptive immunotherapy have been applied clinically. Results of these clinical trials indicate a distinct benefit for select patients. The recent breakthrough of immunologic checkpoint inhibition is being rapidly introduced to a variety of tumor types including sarcoma. It is anticipated that these emerging immunotherapies will exhibit clinical efficacy for a variety of sarcomas. The increasing ability to tailor immunologic therapies to sarcoma patients will undoubtedly generate further enthusiasm and clinical research for this treatment modality. Some of the earliest evidence for antitumor immune reactivity was noted in sarcoma tumors dating well over a hundred years ago. The pioneering work performed by William B. Coley demonstrated the ability of a patient's own immune system to reject multiple metastatic sarcoma tumors by injecting the tumors with a live preparation of streptococcal organisms designated as “Coley's toxin” . Coley nTumor vaccines stimulate an immune response via specific tumor epitopes while increasing activation of tumor specific T-cells and B-cells . VaccineNCT00948961). A similar active study explores the immune response of recombinant NY-ESO-1 antigen and an adjuvant, GLA-SE (NCT02015416).The cancer-testes antigen NY-ESO-1 represents an attractive target in STS vaccine establishment. It is expressed in 80% of synovial sarcomas and nearly all myxoid/round cell liposarcomas , 8. GiveNCT01522820). Inhibition of mTOR has been shown to strongly influence vaccine induced CD8+ T-cell response, leading to greater antitumor efficacy [NCT00005628) in patients with recurrent soft tissue sarcoma are also pending.Another ongoing phase I trial examines the role of recombinant NY-ESO-1 via intranodal injection with or without the mammalian target of rapamycin (mTOR) inhibitor sirolimus in NY-ESO-1 expressing tumors (efficacy . The resNCT01141491). The primary endpoint of PFS revealed no statistically significant difference between treatment groups; however, serologic responses were seen in the vaccine arm versus placebo (98 versus 21%) and minimal toxicities were described [Gangliosides are richly expressed in sarcomas, making them an exceptional target for vaccine therapy. Among them, GM2, GD2, and GD3 are the most abundant , 10. A rt representing the fusion of SYT (at 18q11) with either SSX1 or SSX2 [α was given as an adjuvant agent. While nine patients showed an increase in cytotoxic T-cells, only one patient had a decrease in tumor size [Essentially all synovial sarcomas contain  or SSX2 . SYT-SSXmor size .™) vaccine in patients with Ewing's sarcoma (NCT01061840).Although attempts to use adjuvants such as GM-CSF to increase the immune system response have not yet translated to increased efficacy , 15, an CTLA-4 was the first immune checkpoint receptor to be clinically targeted. Normally after T-cell activation, CTLA-4 is upregulated on the plasma membrane, leading to downregulation of T-cell function through a variety of mechanisms , 17. CTLIpilimumab has demonstrated overall survival in metastatic melanoma with response rates of 10–20% . Data frNCT01643278).In a KIT-mutant GIST mouse model, it was found that the immune system contributed extensively to the antitumor effects of imatinib, a tyrosine kinase inhibitor. Imatinib therapy activated CD8+ T-cells and induced regulatory T-cell apoptosis within the tumor by reducing immunosuppressive enzyme expression . The criThe programmed cell death protein 1 (PD-1) receptor has proven to be an effective immunological target in solid tumor malignancies including non-small-cell lung cancer (NSCLC), melanoma, and renal cell carcinoma (RCC) with response rates exceeding 40% in previously untreated melanomas , 25. ResNCT02301039). The study will also investigate CD8+ T-cells in tumor tissues before and after treatment. Recent data evaluated the role of tumor-infiltrating lymphocytes preexisting in the tumor prior to anti-PD-1 blockade and its predictive value for antitumor response [The role of PD-1/PD-L1 in soft tissue sarcomas is currently being evaluated. An ongoing phase II study (SARC028) is exploring the role of the anti-PD-1 antibody Pembrolizumab in patients with advanced sarcoma. The primary outcome measure is objective response rates with assessments conducted at 8 weeks, up to 5 years. Secondary outcome measures will include adverse events, overall survival, progression-free survival, and response rate by immune-related response criteria . Two other studies explored combination therapy using adoptive T-cell transfer of modified T-cells with biologic therapy, interleukin-2. The study by Mackall et al. combined priming with tumor-peptide pulsed dendritic vaccination followed by T-cell harvest via apheresis for patients with newly metastatic or recurrent high-risk pediatric sarcomas. Patients underwent cytoreductive chemotherapy followed by reinfusion of T-cells and treatment with various doses of interleukin-2. Patients receiving immunotherapy experienced minimal toxicity [NCT01967823), patients with metastatic cancers expressing the NY-ESO-1 antigen undergo a nonmyeloablative lymphodepleting preparative regimen followed by anti-NY-ESO-1 reactive T-cell receptor retroviral vector transduced autologous peripheral blood lymphocytes. Patients subsequently receive up to a maximum of 15 doses of high-dose interleukin-2 therapy. The study is currently recruiting patients.A phase I study examining autologous T-cells and cyclophosphamide in treating patients with metastatic or surgically inoperable myxoid/round cell liposarcoma and synovial sarcoma has been completed (toxicity . In an o in vitro [NCT01898663) combines adenovirus-transfected autologous dendritic cells engineered to express MUC1 and survivin with cytokine-induced killer cells for treatment of intermediate and high-grade sarcomas. Preclinical data in rhabdomyosarcoma showed that survivin-responsive conditionally replicating adenovirus regulated with multiple factors (Surv.m-CRAs) could effectively kill all populations of rhabdomyosarcoma cells, including both stem cells and their progeny [Adoptive transfer strategies also include natural killer cells. This approach already has enthusing preclinical evidence in sarcomas. Twenty-one patients underwent harvesting of cytokine-induced natural killer cells, inhibiting autologous tumor xenograft growth in mice and destroying allogeneic and autologous sarcoma cellsin vitro , 32. An  progeny . In anot progeny . A case  progeny .NCT01953900).Chimeric antigen receptor (CAR) modified T-cell therapy has shown promise in hematologic malignancies . A recenInitial evaluations of the safety and efficacy of CAR T-cells in sarcoma patients set the stage for additional studies that combine adoptive immunotherapy with other immunomodulatory approaches to enhance the expansion and persistence of the T-cells and NK cells. in vivo. The demethylating agent decitabine has been shown to epigenetically modify sarcoma cells, effectively increasing the expression of various antigens and peptides that increase the probability of a targeted T-cell response [One method of optimizing the efficacy of current immunotherapy technologies is through upregulation or modification of tumor cell antigens, increasing their immunogenicityresponse . Pollackresponse . in vitro and inhibited tumor xenograft growth [In another study, pretreatment of a patient-derived primary osteosarcoma cell line with the HDAC inhibitor entinostat led to enhanced overall cytotoxicityt growth , 40. TheNCT01449279, NCT01565837, and NCT01497808). The innovative approach of using combination immunotherapy and radiotherapy as a noninvasive strategy may prove applicable to soft tissue sarcomas in the near future.An abscopal effect has been described anecdotally, in patients with metastatic melanoma where the addition of radiotherapy to ipilimumab led to tumor response in nonradiated lesions in addition to the radiation lesion , 42. In Lastly, major advancements in tumor cell whole exome sequencing (WES) have allowed the identification of endogenous reactive T-cells to previously unknown tumor antigens . ThroughThe historic observations of sarcoma tumors inducing immune responses have matured into feasible and effective immunotherapies in use today. With a greater understanding of the process of T-cell activation , antitum


Corticotropin-releasing factor (CRF) is the hallmark brain peptide triggering the response to stress and mediates—in addition to the stimulation of the hypothalamus-pituitary-adrenal (HPA) axis—other hormonal, behavioral, autonomic and visceral components. Earlier reports indicate that somatostatin-28 injected intracerebroventricularly counteracts the acute stress-induced ACTH and catecholamine release. Mounting evidence now supports that activation of brain somatostatin signaling exerts a broader anti-stress effect by blunting the endocrine, autonomic, behavioral (with a focus on food intake) and visceral gastrointestinal motor responses through the involvement of distinct somatostatin receptor subtypes. The sst2a is the receptor most abundantly expressed at this site compared to other subtypes and microinjection of somatostatin into the RVLM induces a sympatho-inhibitory response mimicked by the sst2 agonist, lanreotide and prevented by an sst2 antagonist, BIM-23627 in rats  involved, icv injection of octreotide  did not alter ic ODT8-SST's action  alter gastrointestinal transit resulting in an inhibitory effect in the upper gastrointestinal tract, while stimulating colonic propulsive motor function , sst2 (S-346-011), or sst4 (S315-297) peptide agonists had no effect  or selective peptide sst2 or sst4 agonists, S-346-011 and S-315-297, respectively did not modify the acute stress-induced stimulation of fecal pellet output , autonomic (sympatho-inhibition) and behavioral (suppression of food intake and anxiety) responses. With regards to the brain-gut axis, the hindbrain sst5 plays a key role in counteracting the stress-induced suppression of gastric emptying, whereas forebrain sst1 reduces the stress-related stimulation of propulsive colonic motor function  have been characterized by the use of selective peptide agonists and antagonists or genetic manipulations in rodents. The sstn Figure , 2000 orn Figure  in attenn Figure . LikewisAll authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The present studies employing Ussing chamber technique identified two opposing short-circuit current (Isc) responses to basolateral bumetanide in rat distal colon. Apart from the transepithelial Cl--secretory Isc in early distal colon that was inhibited by bumetanide, bumetanide also stimulated Isc in late distal colon that had not previously been identified. Since bumetanide inhibits basolateral Na+-K+-2Cl- cotransporter (NKCC) in crypt cells and basolateral K+-Cl- cotransporter (KCC) in surface epithelium, we proposed this stimulatory Isc could represent a KCC-mediated Cl- absorptive current. In support of this hypothesis, ion substitution experiments established Cl- dependency of this absorptive Isc and transport inhibitor studies demonstrated the involvement of an apical Cl- conductance. Current distribution and RNA sequencing analyses revealed that this Cl- absorptive Isc is closely associated with epithelial Na+ channel (ENaC) but is not dependent on ENaC activity. Thus, inhibition of ENaC by 10 μM amiloride or benzamil neither altered the direction nor its activity. Physiological studies suggested that this Cl- absorptive Isc senses dietary Cl- content; thus when dietary Cl- was low, Cl- absorptive Isc was up-regulated. In contrast, when dietary Cl- was increased, Cl- absorptive Isc was down-regulated. We conclude that an active Cl- extrusion mechanism exists in ENaC-expressing late distal colon and likely operates in parallel with ENaC to facilitate NaCl absorption.Mammalian colonic epithelia consist of cells that are capable of both absorbing and secreting Cl Under normal non-diarrheal conditions, [Cl-]L in the colon is low because, under these conditions, the main luminal anions are short-chain fatty acids (SCFAs), which may have a concentration as high as 120 mM [-]L falls further (due to reabsorption), and when it reaches the distal colon, particularly the late distal colon, the [Cl-]L can become extremely low . Thersc in late distal colon. This is because ENaC-mediated electrogenic Na+ absorption is restricted to the surface epithelium in this portion of the colon .Under the present experimental conditions, the changes in Ilsewhere –13 and tnsporter , 14. A p--free, HCO3--free, or Na+-free Ringer solutions. In those experiments, tissues were bathed initially in normal Ringer solutions and then in respective Cl-/HCO3-/Na+-free Ringer solutions, as described i) must fall appreciably. In the distal colon, while Ψa depolarizes due to ENaC-mediated electrogenic Na+ absorption, it also hyperpolarizes due to spontaneous electrogenic K+ secretion. Furthermore, unlike exclusive absorptive epithelia , colonic epithelia have a more complex task of providing both absorption and secretion of Cl-, and [Cl-]i may or may not fall in this tissue due to the presence of Cl- entry into the cell from the basolateral NKCC1 [-/ HCO3- exchanger [a and [Cl-]i are sufficient to maintain the driving force for Cl- influx from the lumen, requisite for passive Cl- absorption to occur in a low [Cl-]L situation. It is likely that an alternative active mechanism for Cl- absorption is present in the distal colon, particularly in the late distal colon where, due to reabsorption, [Cl-]L is the lowest i) and elevates intracellular K+ concentration ([K+]i), generating two chemical gradients across cell membrane: the gradient for Na+ and the gradient for K+. Whereas the gradient for Na+ enables apical ENaC to move Na+ from the lumen into the cell, the gradient for K+ provides the driving force for active uptake of Cl- via the secondary active basolateral bumetanide-sensitive KCl cotransporter i; the apical NPPB/glibenclamide-sensitive Cl- conductance (likely a Cl- channel/transporter) subsequently moves Cl- from the lumen into the cell.According to this new model, Nansporter  (likely nsporter ), which - in this part of the colon. Other transporters may be involved to facilitate this transport process. These may include: 1) basolateral K+ channel. This K+ channel may participate in the fine-tuning of the activity of KCC1 through adjusting [K+]i; 2) basolateral Cl-/ HCO3- exchange. There are reports that this anion exchange may contribute to Cl- secretion L is low; 4) apical DRA [-/HCO3- exchanger is reported to have concurrent conductive Cl- activity [- from the lumen when [Cl-]L is low through using the gradient of HCO3- generated internally through the hydration of CO2 by carbonic anhydrase  and CaSR activity decrease electrogenic Cl- secretion [2+] is stable without significant elevations. Whether CaSR increases electrogenic Na+ and Cl- absorption in this highly regulated late distal colon remains to be determined.Like ENaC, this active Clated see . In contated see . In respand CHIF . Whethereostasis –36. Increcretion , 37, 38;ecretion , 38. The+ movement is secretory  in the proximal colon and is electroneutral (via NHE3) and electrogenic (via ENaC) in the distal colon [+ transport in the proximal colon [+ transport from NHE3 to ENaC in the distal colon [+ depletion and a significant decrease  in response to dietary Na+ repletion  in the +-ATPase ) in the al colon . Similaral colon , 42. Aldal colon , but caual colon . Consistepletion . It woul- secretory Isc, which, in current direction, is inhibitory by bumetanide, was primarily localized to the salt secretory early distal colon, whereas the Cl--absorptive Isc, which, in current direction, is stimulatory by bumetanide, was primarily present in the salt absorptive late distal colon  active Cl- absorption in the late distal colon is increased by dietary Cl-  depletion and decreased by dietary Cl-  repletion; and 3) active Cl- absorption occurs through transcellular transport that involves an apical NPPB/glibenclamide-sensitive Cl- conductance and a basolateral bumetanide-sensitive K+-Cl- cotransporter. We conclude that an electrogenic Cl- extrusion mechanism exists and operates in parallel with the electrogenic ENaC in the distal colon (and probably in nephron as well) where the final control of NaCl absorption occurs before effluents are discharged from the body.In summary, our present studies demonstrate the following: 1) rat distal colonic epithelium is able to both actively secrete and absorb Cl


Salmonella enterica, resides within the Salmonella-containing vacuole (SCV) inside epithelial cells and macrophages. Salmonella hijacks the host endosomal system to establish this unique intracellular replicative niche, forming a highly complex and dynamic network of Salmonella-induced filaments (SIFs). SIFs radiate outwards from the SCV upon onset of bacterial replication. SIF biogenesis is dependent on the activity of bacterial effector proteins secreted by the Salmonella-pathogenicity island-2 (SPI-2) encoded type III secretion system. While the presence of SIFs has been known for almost 25 years, their precise role during infection remains elusive. This review summarizes our current knowledge of SCV maturation and SIF biogenesis, and recent advances in our understanding of the role of SIFs inside cells.A common strategy among intracellular bacterial pathogens is to enter into a vacuolar environment upon host cell invasion. One such pathogen,  Salmonella enterica serovars are Gram-negative bacterial pathogens capable of causing enteric disease in all vertebrates. S. enterica serovars are associated with illnesses ranging from gastroenteritis to typhoid fever caused by non-typhoidal Salmonella (NTS) serovars and S. enterica subsp. enterica serovar Typhi, respectively  following host cell invasion. Entry into a bacteria-containing vacuole (BCV) is seen in diverse intracellular pathogens including Legionella pneumophila, Shigella flexneri, Francisella tularensis, Mycobacterium tuberculosis, and Edwardsiella spp  escape the SCV and enter a hyper-replicative state within the cytosol of epithelial cells  radiating outwards from the SCV, throughout the host cell's cytoplasm. Salmonella-induced filaments (SIFs) are the most abundant, and best studied, form of SIT. Intravacuolar replication accompanies formation of SIFs which are endosomal-tubule extensions characterized by the presence of lysosomal glycoproteins and other endocytic markers  tubules (SISTs) . While SPI1-T3SS-secreted effectors enable host cell invasion and SCV biogenesis, the SPI2-T3SS-secreted effectors are associated with SCV maturation, SIF biogenesis, and promoting survival and replication  in HeLa cells resemble early endosomes with markers for endocytic sorting and recycling pathways and subsequent maturation pathways, partially owing to the activities of T3SS-secreted effectors. The SPI1-T3SS-secreted effector SopB maintains high levels of SCV membrane-associated phosphatidyl-3-phosphate during initial stages of SCV maturation  1, 2, and 3, and vATPase ; the SifA-SKIP complex binds and sequesters Rab9, inhibiting Rab9-dependent M6PR recruitment to the SCV membrane. Decreased M6PR recruitment to the SCV membrane decreases recruitment of lysosomal enzymes to the SCV, thereby protecting intracellular Salmonella replication coincides with full maturation of the late SCV and extension of SIFs. The remarkably dynamic process of SIF biogenesis results in a highly complex stabilized network of SIFs by 8 h.p.i., during which individual SIFs undergo extension, contraction, branching, and fusion with other SIFs  of HeLa cells. The same SPI2-T3SS-secreted effectors associated with SCV maturation are also associated with SIF biogenesis. These effectors are SifA, SseJ, SopD2, PipB2, SseF, SseG, SpvB, and SteA Table . All eigsifA mutants fail to induce SIFs in HeLa cells , while remaining separated from host cell cytosol . This model accounts for the activities of four of the eight SPI2-T3SS-secreted effectors associated with SIF biogenesis, namely SifA, PipB2, SseF, and SseG. It remains unknown how the other four effectors contribute in this model.Krieger et al. propose a model of SIF biogenesis wherein SPI2-T3SS-secreted effectors, in particular SifA, recruit and fuse host membrane vesicles to the SCV providing components for tubule extension. PipB2 then promotes SIF extension by linking nascent SIFs to the kinesin-1 molecular motor promoting extension of SIFs outwards from the SCV along microtubules  is also a major component of SIFs, which unlike most SIF markers is not associated with late endocytic compartments  and controls multivesicular endosome biogenesis in uninfected cells , there is the potential that the SCV associates with the ER system via multiple contact sites (MCSs). MCSs are regions where two organelles are in close apposition (between 3 and 15 nm at ER MCSs) and permit communication between organelles  SIFs collect host membrane components providing access to endocytosed compounds, including nutrients, enabling replication and membrane expansion; and (2) the SCV/SIF interconnectivity reduces exposure of intravacuolar Salmonella to the host defenses by diluting lysosomal enzymes inevitably acquired by the SCV. Fusion of host endocytic cargo from various intracellular locations to provide the membrane materials enabling SIF biogenesis explains both the abundance and variety of host markers found within SIF and SCV membranes.The ability to form BCVs, survive, and replicate within host cells is a common strategy among intracellular pathogens. Unique to Salmonella's intracellular lifestyle, additional work is needed to characterize SIFs if we wish to fully understand S. Typhimurium's intracellular niche. Despite the large body of research regarding SIFs, several areas require further investigation. It remains unclear why S. Typhimurium has evolved to form complex multi-SPI2-T3SS-effector-dependent SIFs. The mechanisms underpinning SIF biogenesis still requires work to determine how all eight implicated SPI2-T3SS effectors create these unique structures. The ability to form SIFs correlates with the ability to cause disease in mouse models of infection (Stein et al., Salmonella in association with LAMPs in infected tissues (Zhang et al., Salmonella's unique replicative niche, and may reveal novel insights into other intracellular pathogens.Although recent studies have significantly advanced our understanding of KK drafted the manuscript; BF revised the manuscript critically for important intellectual content.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


P < 0.05). Multivariate analysis showed that the pre-post-CEA ratio is associated with lymphatic and distant metastasis, tumor-node-metastasis (TNM) stage and degree of tumor differentiation . The pre-CEA levels, pre-post-CEA ratios, distant metastasis, TNM stage and degree of tumor differentiation were all associated with 5-yr overall survival  based on multivariate analysis. Consequently, pre-CEA levels, pre-post-CEA ratios, distant metastasis and TNM stage are independent risk factors for CRC. We have thus demonstrated that the pre-post-CEA ratio is a prognostic indicator for CRC patients.We explored the prognostic significance of the pre-operative-to-post-operative serum carcinoembryonic antigen (pre-post-CEA) ratio in colorectal cancer (CRC). We detected pre- and post-operative CEA levels in 2035 CRC patients surgically treated at First Affiliated Hospital of Zhengzhou University between June 2001 and June 2011. Univariate analysis revealed the pre-post-CEA ratio is associated with distant metastasis and degree of tumor differentiation (both  Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the fourth leading cause of cancer death worldwide with steadily increasing mortality rates , 2. In ret al. reported that the pre-post-CEA ratio is prognostic predictor after surgery for stage III rectal cancer patients [In recent years, tumor biomarkers have been widely used in clinical diagnosis, post-operative monitoring of tumor recurrence, prognosis and curative therapy of CRC patients . The serpatients . HoweverAfter screening 2833 CRC patients, we excluded the following: 1) unresectable (628 cases); (2) combined with other cancers (27 cases); (3) benign disease (94 cases); (4) pregnancy (2 cases); (5) emergency (24 cases) and (6 cases) untreatable due to widespread metastasis (23 cases). Finally, we enrolled 2035 patients including 1138 males and 897 females; of these, 872 had colon cancer and 1163 had rectal cancer .The patients were divided into low and high expression groups based on pre-CEA levels and pre-post-CEA ratios based on their mean values. Univariate analysis showed that pre-CEA levels were associated with smoking, tumor size, lymphatic and distant metastases and TNM stage . Meanwhile, pre-post-CEA ratios were associated with lymphatic and distant metastases, TNM stage and degree of tumor differentiation .Multivariate analysis showed that pre-CEA levels were associated with family history, smoking, tumor size, distant metastasis and TNM stages . Factors like age, gender, family history, smoking, tissue type and tumor location were not related with 5-year OS .As shown in Table P < 0.05, Table P > 0.05, Table Multivariate survival analysis showed that pre-operative CEA, pre- to post- operative CEA ratio, distant metastasis, TNM stage and degree of tumor differentiation were associated with 5-year OS . Also, patients in groups 3 and 4 showed lower OS than group 1 patients and higher OS than group 2 patients  represents a group of macromolecular acid glycoproteins expressed highly during fetal development and reduced in the adults , 15. Higet al. also reported that elevated pre- and post-operative serum CEA levels were associated with recurrence and poor clinical outcomes in few CRC patients [et al showed that high serum CEA or CYFRA 21-1 levels before and after surgery were associated with poor overall survival (OS) and progress-free survival (PFS) in stage I non-small cell lung cancer [Many studies have suggested that CEA levels can be used for predicting metastasis although few contradictory reports exist –20. Highpatients . Duan etg cancer . In thisLymph node metastasis  and distIn previous studies, pre-CEA levels were identified as reliable indicators of prognosis and therapeutic efficacy in stage II and III CRC patients , 29. ManApart from the prognostic value of pre-CEA levels, few studies have also reported the prognostic value of pre-operative and post-operative CEA levels in combination . TherefoBased on tumor source, the degree of malignancy determined by histology has been used to classify different pathological types of tumors , 33. MosDuring malignant transformation of CRC cells, overexpression of CEA lowers the function of other adhesion molecules and interferes in the interaction between normal cells, thereby weakening the single-layer structure of glandular tube –35, 36. There is consensus that lymphatic metastasis, distant metastasis and TNM stage dictate prognostic status of CRC patients. In our study, these three indicators were also independent risk factors in relation to for pre-post-CEA ratios. Therefore, pre-post-CEA ratios are an essential risk factor for prognosis of CRC patients.In conclusion, pre-post-CEA ratios were associated with the malignant phenotype of lymphatic metastasis, distant metastasis, TNM stage and degree of tumor differentiation in CRC patients. It was also an independent risk factor for CRC patients. Therefore, we postulate that pre-post-CEA ratio is a potential tumor biomarker to evaluate prognosis and therapeutic efficacy in CRC patients.This study was approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University (No.ZZUF-2016174). All patients provided informed written consent prior to their participation. Clinicopathological parameters and follow-up data were obtained 2035 CRC patients  that received radical surgery in the First Affiliated Hospital of Zhengzhou University between June 2001 and June 2011. The mean patient age was 59.84+13.44 years. Among the 2035 CRC patients, 872 had colon cancer and 1163 rectal cancers. Colon cancer diagnosis was confirmed by histopathology prior to their participation. None of the enrolled patients received pre-operative chemotherapy or immunotherapy. Tumor-node-metastasis (TNM) stage was determined according to the American Joint Committee on Cancer/International Union Against Cancer TNM staging system of colorectal cancer .Blood samples (4ml each) were obtained within the week before surgery for preoperative CEA estimation and 1–2 weeks after the surgery for post-operative CEA estimation. Serum CEA levels were estimated by ELISA kit  according to manufacturer's instructions.After surgery, the patients were assessed once a month for the first 6 months, once every 3 months for the first 2 years, once every 6 months until 5 years followed by once a year. Follow-ups were either by outpatient or inpatient review or by telephone. 216 patients did not participate in the follow-up analyses because they did not communicate with the physicians after surgery. In addition, 13 patients developed dysthymia and were unable to cooperate with the study; 3 patients committed suicide and 19 patients did not participate in the follow-up for unknown reasons. Therefore, the total follow-up rate in the study was 87.67%.FOLFOX6 was used as the first line adjuvant or neoadjuvant therapy scheme for CRC patients with high-risk IInd or IIIrd stage. CapeOX was used as the chemotherapy scheme as either 1st or 2nd line adjuvant or neoadjuvant therapy for CRC patients with high-risk IInd, IIIrd or IVth stage, drug resistance or postoperative recurrence. FOLFIRI was used as the chemotherapy scheme for CRC patients in advanced IVth stage, postoperative recurrence, metastasis or drug resistance.Radical surgery was performed according to complete mesocolic excision (CME) for colon cancer patients and total mesorectal excision (TME) principle for rectal cancer patients.2 test to analyze pre-CEA levels and pre-post-CEA ratios and clinicopathological parameters. Kaplan Meier survival curves and log-rank test were used to compare the low and high level groups of pre-CEA levels and pre-post-CEA ratios. Multivariate survival analysis was performed by the Cox regression model to determine relative risk (RR) and 95% confidence intervals (CI). Statistical significance was defined as P < 0.05.All statistical analyses were carried out using SPSS for Windows version 20 . Univariate analysis was performed by χ