Eukaryotic polycistronic transcription units are rare and only a few examples are known, mostly being the outcome of serendipitous discovery. We claim that nonsense-mediated mRNA decay (NMD) immune structure is a common characteristic of polycistronic transcripts, and that this immunity is an emergent property derived from all functional CDSs. The human RefSeq transcriptome was computationally screened for transcripts capable of eliciting NMD, and which contain an additional ORF(s) potentially capable of rescuing the transcript from NMD. Transcripts were further analyzed implementing domain-based strategies in order to estimate the potential of the candidate ORF to encode a functional protein. Consequently, we predict the existence of forty nine novel polycistronic transcripts.Experimental verification was carried out utilizing two different types of analyses. First, five Gene Expression Omnibus (GEO) datasets from published NMD-inhibition studies were used, aiming to explore whether a given mRNA is indeed insensitive to NMD. All known bicistronic transcripts and eleven out of the twelve predicted genes that were analyzed, displayed NMD insensitivity using various NMD inhibitors. For three genes, a mixed expression pattern was observed presenting both NMD sensitivity and insensitivity in different cell types. Second, we used published global translation initiation sequencing data from HEK293 cells to verify the existence of translation initiation sites in our predicted polycistronic genes. In five of our genes, the predicted rescuing uORFs are indeed identified as translation initiation sites, and in two additional genes, one of two predicted rescuing uORF is verified. These results validate our computational analysis and reinforce the possibility that NMD-immune architecture is a parameter by which polycistronic genes can be identified. Moreover, we present evidence for NMD-mediated regulation controlling the production of one or more proteins encoded in the polycistronic transcript. The vast majority of eukaryotic genes are considered monocistronic with a single transcription unit encoding for a single protein . Polycistronic transcription units  are rare in eukaryotes and specifically in mammals, and therefore little is known on how they differ from the monocistronic ones.Genomically organized polycistronic units are known in several organisms  yet those are trans-spliced and each monocistronic unit is translated independently Newly synthesized mRNAs are subjected to a pioneer round of translation in which premature termination codon (PTC) containing transcripts are identified and degraded in various degrees of efficiency via the Nonsense-mediated mRNA decay (NMD) mechanism Seven polypeptides constitute the mammalian NMD core mechanism: up-frameshift protein 1 (UPF1), UPF2, UPF3 (comprised isoforms UPF3 and UPF3X) SMG1, SMG5, SMG6 and SMG7. UPF1 is the most conserved, essential protein, with RNA-dependent ATPase and 5′-3′ helicase activities Turning a polycistronic transcript into NMD-immune following the pioneer round of translation requires the removal of all EJCs subsequent to the translation of the functional CDS. In this manuscript we raise the hypothesis that human  functional polycistronic transcripts share a distinctive NMD-immune architecture, which is an emergent property of all functional CDSs. Further, we argue that the definition of potentially NMD-eliciting transcripts (failing to fulfill the "55 nucleotide rule") ought to include the 5' UTR of the molecule, as occurs in many upstream open reading frame (uORF) containing transcripts. In non-polycistronic transcripts. uORFs, which are found in almost 50% of human genes, are mainly characterized by their negative regulatory effect A computational-based approach was utilized to survey monocistronic and polycistronic transcript architecture and to predict the existence of novel polycistronic transcripts in the human transcriptome. We screened the human RefSeq dataset for potentially NMD-eliciting transcripts, according to the classic definition and our modified one. Further, we aimed to isolate those transcripts containing ORFs capable of "rescuing" the mRNA from its NMD-eliciting destiny, i.e., overlapping the exon junctions or positioned in their proximity (as detailed in the Methods section). We then applied domain-based strategies (see below) to predict the potential of the candidate ORF to encode a functional protein. Polycistronic (mainly bicistronic) transcript prediction is presented and discussed.Our main hypothesis was that polycistronic transcripts share a distinctive NMD-immune architecture, leading to the production of stable mRNA, maximally available for translation. We thus assessed the architecture of the known human polycistronic and bicistronic transcripts . In two per se seem to be inadequate based on the lack of evolutionary conservation in the known bicistronic genes.Based on the architecture of the known bicistronic transcripts, we devised a strategy for the identification of novel polycistronic genes. Polycistronic transcript search was limited to potentially NMD-eliciting transcripts with an annotated stop codon positioned in the penultimate or upstream exon. Transcripts in which the annotated stop codon is positioned in the terminal exon (similar to the MTPN-LUZP6 gene) were excluded from this study due to the following reasons: (i) the vast majority of the known mammalian bicistronic genes share an NMD-immune architecture contributed by all functional CDSs; (ii) no other criteria were indicative enough: our preliminary results show that ORF coding potential alone is insufficient to distinguish functional ORFs from non-functional ones (data not shown). Furthermore, comparative genomics In all, 30035 Refseq records were analyzed for potentially failing to fulfill the “55 nucleotides rule” and eliciting NMD, as detailed in the Methods section. Of these, 113 transcripts contained an annotated stop codon positioned 55 nucleotides or more upstream to the terminal exon-exon junction. Those were further analyzed for the existence of ORFs which are potentially capable of turning the transcript from NMD-eliciting into NMD-immune. Ninety three potential rescuing ORFs were identified in 68 transcripts.in silico identification is problematic, and most studies focus on empirical data validation not on novel IRES prediction The existence of a rescuing ORF overlapping an exon junction is far from sufficient in order to identify a polycistronic transcript. We therefore assessed potential functional ORFs based on the following criteria: (i) Existence of a translation initiation sequence. Two potential elements - Kozak-like sequence and internal ribosome entry sites (IRESs) in the 5' end can be considered. We avoided relying on IRES identification as a search criterion because its presence in cellular mRNAs is still debated Out of the 93 potential rescuing ORFs, 53 (39 transcripts) were discarded due to high homology between the rescuing ORF and the annotated CDS. The remaining ORFs were further analyzed according to the criteria elaborated above. Eight candidate bicistronic transcripts (6 genes) were identified, out of which two were discarded because the predicted protein was identified to contain only a signal peptide sequence, with no other known protein domains (See Methods section). From the remaining six transcripts, three novel (2 genes) and three known bicistronic transcripts  were identified . Similar to the strategy undertaken in the former stage, we needed to distinguish transcripts which contain a regulatory uORF from polycistronic ones in which the upstream CDS is still unknown. The upstream CDSs in polycistronic transcripts and regulatory uORFs differ first and foremost by their NMD-induction potential. Thus we performed a preliminary analysis aiming to identify potentially NMD-eliciting transcripts based on mRNA 5' screening. We analyzed the distribution of the annotated ATG exon position in human RefSeq transcripts and evaluated how many of them are potentially NMD-eliciting . NMD degradation induction relies on EJCs that remain after the pioneer round of translation. Since no known sequence-based parameters are available to indicate whether translation re-initiation will occur in sequential ORFs, our approach is applicable only for those cases in which the uORF/CDS and the annotated ATG are positioned in different exons and therefore at least one remaining EJC potentially exists. Transcripts for which the first exon contains the 5′ UTR and the annotated ATG, as well as potentially encoding ORF, were not included in our study as they require experimental evaluation of re-initiation and NMD-eliciting potential. We found that only 59% of the annotated ATGs are positioned in the first exon of the transcript and the rest are positioned in the second or downstream exons .Transcripts in which the annotated ATG is positioned in the second or downstream exons were analyzed for 5′ UTR ORF existence . Of these, 6118 transcripts  contain no ORF in their 5′ UTR, i.e., the ribosomal 43S pre-initiation complex is assumed to scan the mRNA until the annotated ATG is reached (detaching pre-deposited EJCs on its way) At this point we concluded the following: (i) a considerable portion of RefSeq transcripts contain two or more ORFs, are in NMD-immune architecture, and therefore have the potential to function as polycistronic mRNAs; (ii) NMD-eliciting potential in the human transcriptome is likely higher then so far evaluated, due to 5′ UTR-related NMD-eliciting architecture .After dividing the transcriptome into groups according to the annotated ATG position and the existence of rescuing uORFs, we turned to predict the 5' UTR-related novel polycistronic transcript potential. A total of 4130 transcripts (13.8% of Refseq transcriptome) constitute the dataset from which we aimed to differentiate transcripts with regulatory uORFs from those with functional upstream CDSs.Two working assumptions guided this stage: (i) the first ATG identified by the 43S pre-initiation complex can be positioned in the second and downstream exon, and all EJCs deposited upstream to it are removed. Therefore no full exon-junctions coverage is required, and instead we screened for exon-junction coverage between the end of the first ORF identified and the annotated ATG. (ii) potential ORFs were analyzed only if the ORF was larger than 99 nucleotides. This cutoff value was set based on the size range of known polycistronic encoded proteins  and the relatively small size of human uORFs (average length 51.5 nucleotides) We hypothesized that human polycistronic mRNAs share a unique configuration, in which functional CDSs are mutually organizes in an NMD-immune structure. This architecture was demonstrated in four known bicistronic genes , and was further used to predict the existence of 49 novel polycistronic transcripts. In order to validate our predictions, we screened the literature and databases for known cases of NMD inhibition and transcription initiation site detection.mRNA expression datasets from Gene Identification by NMD inhibition (GINI) experiments, in which mRNA levels are compared in the presence and absence of NMD- inhibitors  were utilized to identify polycistronic transcript NMD sensitivity. Five datasets representing a variety of cell types were downloaded from the Gene Expression Omnibus database (GEO) and analyzed. per se is not sufficient, as some transcripts may show detectable levels of seemingly stable mRNA even though NMD degradation does occur. A more direct verification is therefore desired, reinforcing our validation results. We therefore crosschecked our predicted transcript list with a dataset of experimentally verified TISs, produced by utilizing global translation initiation sequencing (GTI-seq) in the transcriptome of human embryonic cell line (HEK293) et al. TIS dataset, in the exact position and with the same ORF size as predicted in our study  share a unique configuration, in which functional CDSs are mutually organized in an NMD-immune architecture. Indeed, we detected NMD-immune transcript architecture in the known human bicistronic genes. Further, we analyzed the human Refseq transcriptome, predicting the existence of 49 novel polycistronic transcripts. Bioinformatics-based analysis, indicating the existence of known protein domains in the predicted functional-ORFs encoded proteins or their similarity to known proteins, supports our assumption regarding the encoded proteins' potential to be produced and active. mRNA expression datasets from GINI experiments were utilized for experimental verification, aiming to explore whether the allegedly polycistronic transcripts are insensitive to NMD. All the known bicistronic transcripts and an additional eleven genes from our predicted gene list displayed NMD insensitivity for various NMD inhibitors, including emetine, caffeine and UPF1 and Ago2 siRNAs (other genes in our list were not sampled in those GINI experiments). The gene ZNF481, which was studied in a single experiment, displayed an NMD sensitive profile. An additional three of the eleven genes displayed a mixed expression pattern of both NMD sensitivity and insensitivity in different cell types, possibly manifesting time-dependent and process-dependent translation re-initiation regulation. Furthermore, some of the genes displayed significantly high levels of expression which do not fit the expression pattern of inefficient NMD. Hence, these results reinforce our view that an NMD-immune architecture is likely to play a role in polycistronic transcript expression regulation. Finally, we succeeded to find 9 predicted polycistronic transcripts (5 genes) in a translation initiation sites dataset, with the exact position and ORF size as predicted in our study  The rationale for novel polycistronic transcript prediction was based on distinguishing functional CDSs from regulatory ORFs by their potential to elicit NMD and to encode a functional protein. Many studies addressed the question of parameters affecting translation re-initiation following a uORF, including uORF size, length and lack of secondary structure of the intercistronic spacing sequences and the usage of conserved uATG It was shown that following translation of a uORF and the release of the 60S subunit, the 40S subunit may remain on the mRNA and resume scanning for as far as 600 nucleotides, without re-initiating translation Almost half of the human genes have uORFs in their 5′ UTRs, capable of reducing protein expression by 30 to 80% Moreover, we further argue that while evaluating the potential for NMD, both sides of the exon-exon junction (upstream and downstream) ought to be equally considered. EJCs deposited 20–24 nucleotides upstream to the exon junction are being pushed away and removed by the ribosome. The ribosome's spatial dimensions dictate a downstream EJC displacement even if the stop codon is positioned 50 to 55 nucleotides upstream to the exon junction. Based on this fact, we argue that in the event of translation re-initiation, EJC removal is likely to occur even if the ORF starts in close proximity downstream to the exon-exon junction, further changing NMD fraction size as estimated computationally. Indeed, yeast ribosome footprint experiments indicated the protection of 28 nucleotides upstream and downstream to the ATG (−12 to +15 nucleotides) We assume that many functional-ORF encoded proteins are expressed in low levels or to a limited period of time, thus their detection is challenging. Moreover, we believe that seemingly NMD-eliciting, polycistronic transcripts are underrepresented in the mammalian genome annotation, partially due to the tendency to suppress NMD candidate mRNAs from nucleic databases . NMD-eliciting transcripts seem to include remarkable regulatory features, waiting to be further studied. We therefore hope that our hypothesis will be further verified by experimentalists.http://www.ncbi.nlm.nih.gov/RefSeq/) 35157 Human Refseq mRNAs records were downloaded from the RefSeq database  from 5' to 3' direction. In order for an ORF to be defined as a rescuing one, all exon-exon junctions, downstream to the annotated stop codon, are to be covered according to the "55 nucleotide rule". Namely, the ORF should cover the entire sequence length within the range of 55 nucleotides upstream to the first exon-exon junction-downstream to the annotated stop codon, and up to at least 55 nucleotides upstream to the terminal junction (or further downstream). For NMD-eliciting transcripts harboring an annotated stop codon in the penultimate exon in a distance >55 nucleotides, a minimal rescuing ORF should start and end in the penultimate exon in a distance smaller than 55 nucleotides upstream to the terminal exon. If more than one ORF was required in order to turn NMD-eliciting transcript into NMD-immune, they were considered as one unit.Out of 113 NMD-eliciting transcripts, 68 NMD-immune transcripts contained one or more rescuing ORFs . At this stage, all rescuing ORFs were considered, including ORFs which overlap one another in different or identical reading-frames. No overlap was allowed between the annotated CDS and rescuing ORFs.Functional uORFs were searched for in the 5′ UTRs of RefSeq records for which the annotated CDS start codon is in the second or later exon. An ORF screen was carried out in the three reading-frames  from 5' to 3'. Records which followed our hypothesized polycistronic architecture, namely, exon-junctions coverage between the end of the first ORF identified and the annotated ATG and potential ORFs larger than 99 nucleotides, were further analyzed. Out of 4130 transcripts containing ORFs in their 5' UTR with an NMD-immune architecture, 335 transcripts were identified to contain ORFs larger than 99 nucleotides and with no significant similarity between the candidate ORF sequence and the annotated CDS, and were further evaluated for polycistronic and bicistronic candidacy.Initiation of translation by eukaryotic ribosomes is optimal at the ACCATGG consensus sequence BLASTN analysis was carried in order to rule out candidate ORF encoded proteins with significant similarity to the annotated protein of the transcript. ORF sequences were analyzed against the human RefSeq mRNA dataset utilizing BLASTN standalone application with default parameters Candidate functional ORFs were predicted based on whether the potential ORF encoded protein shares a significant similarity to other proteins in the protein database. Candidate ORFs were translated to potential protein sequences and were analyzed for protein similarity utilizing BLASTP  with default parameters against the non-redundant protein sequences (nr) database http://www.ebi.ac.uk/Tools/InterProScan/) Further, candidate functional ORFs were predicted based on whether the potential ORF contains functional domains according to InterProScan analysis. The ORF-encoded proteins were screened for known protein domains utilizing InerProScan Web Services  in which mRNA levels are compared in the presence and absence of NMD- inhibitors of different sorts Click here for additional data file.Table S2(PDF)Click here for additional data file.


Lurcher (Lc/+) and tambaleante (tbl/tbl) mice as models of Purkinje-cell-devoid cerebellum.The cerebellum plays a key role in the acquisition and execution of motor tasks whose physiological foundations were postulated on Purkinje cells' long-term depression (LTD). Numerous research efforts have been focused on understanding the cerebellum as a site of learning and/or memory storage. However, the controversy on which part of the cerebellum participates in motor learning, and how the process takes place, remains unsolved. In fact, it has been suggested that cerebellar cortex, deep cerebellar nuclei, and/or their combination with some brain structures other than the cerebellum are responsible for motor learning. Different experimental approaches have been used to tackle this question . One of these approaches is the study of spontaneous mutations affecting the cerebellar cortex and depriving it of its main input–output organizer . In this review, we discuss the results obtained in our laboratory in motor learning of both  This mutation affects cerebellar Purkinje cells, leading to their disappearance  mutation, the degeneration of Purkinje cells begins from the second month of life—a date from which the Purkinje cell number decreases dramatically. When the tbl/tbl mouse is 1 year old, fewer than 1% of the cerebellar Purkinje cells survive  mutation develops an ataxic syndrome . The diminution on Purkinje cell inhibitory input leads to changes in these neuronal populations that have been closely related to mutant behavioral phenotypes. Among these neural responses, an increase in the parvalbumin (Parv) was consistently found in cerebellar and vestibular nuclei after spontaneous or surgical Purkinje cell deprivation (Grüsser-Cornehls and Bäurle,  Figures . Parv ims Figure —there iss Figure . Therefotbl/tbl mice seemed to perform the horizontal bar test more easily than did Lc/+ ones (Figure tbl/tbl mice were consistently unable to successfully perform the vertical pole test, while Lc/+ mice did (Figure tbl/tbl is the only mutation that losses all Purkinje cells once the cerebellar circuits have developed normally (Wassef et al., tbl/tbl cerebellum regarding the total amount of granule cells after Purkinje cell loss, and the cerebellar cortico-nuclear relationships at the beginning of the mutation effect, would explain these motor learning differences. Accordingly, the comparison of amounts of GABAergic input to cerebellar deep nuclei between Lc/+ and tbl/tbl mice could help to solve the question (see Grüsser-Cornehls and Bäurle, tbl/tbl brain areas involved in motor behavior and the possible compensatory processes taking place after the loss of Purkinje cells when all motor circuitry is fully developed would explain the differences in tbl/tbl motor behavior.Despite the similarities in the structural changes suffered by the different Purkinje-cell-devoid mutant mice sharing a similar motor behavior Table , there as Figure . In contd Figure . These dVarious studies give the cerebellar cortex an important role in motor learning. However, the results obtained in our laboratory, using two Purkinje-cell-deprived mutant mice, show that while this structure is not essential in learning, its absence disturbs the performance and the magnitude of the learned response (Porras-García et al., 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.


Infectious pulmonary complications limit the success of hematopoietic stem cell transplantation (HSCT) as a therapy for malignant and non-malignant disorders. Susceptibility to pathogens in both autologous and allogeneic HSCT recipients persists despite successful immune reconstitution. As studying the causal effects of these immune defects in the human population can be limiting, a bone marrow transplant (BMT) mouse model can be used to understand the defect in mounting a productive innate immune response post-transplantation. When syngeneic BMT is performed, this system allows the study of BMT-induced alterations in innate immune cell function that are independent of the confounding effects of immunosuppressive therapy and graft-versus-host disease. Studies from several laboratories, including our own show that pulmonary susceptibility to bacterial infections post-BMT are largely due to alterations in the lung alveolar macrophages. Changes in these cells post-BMT include cytokine and eicosanoid dysregulations, scavenger receptor alterations, changes in micro RNA profiles, and alterations in intracellular signaling molecules that limit bacterial phagocytosis and killing. The changes that occur highlight mechanisms that promote susceptibility to infections commonly afflicting HSCT recipients and provide insight into therapeutic targets that may improve patient outcomes post-HSCT. Early observations made in the 1950s paved the way for the development of hematopoietic stem cell transplantation (HSCT). Lorenz  showed tPrior to receiving a graft, transplant recipients undergo conditioning regimens that include total body irradiation (TBI), chemotherapy, or a combination of TBI and chemotherapy Aschan, . These rTraditionally, HSCs were aspirated from the bone marrow of donor patients and later infused into recipients. This procedure was often painful for the donor. However, technical advances now allow for mobilization of HSCs into the peripheral blood with the use of the growth factor, granulocyte-colony stimulating factor (G-CSF) Cutler, . TreatmeCandida pseudotropicalis as well as phagocytosis and killing of bacteria including Staphylococcus aureus and Listeria monocytogenes. This same study suggested that the defects may be primarily confined to the lung as the function of peripheral blood monocytes in these same assays was similar to control patients , diffuse alveolar hemorrhage (DAH), idiopathic pneumonia syndrome (IPS), and bronchiolitis obliterans syndrome (BOS) Soubani, . These oCytomegalovirus (CMV) infections predominate in the late phase of engraftment and are associated with increased mortality  is the most common pathogen isolated within 100 days post-engraftment and is associated with high recurrence rates due to increasing antibiotic resistance  models for both syngeneic  and allogeneic  transplantation offer the opportunity to study changes in the pulmonary immune system and microenvironment following transplantation. In these systems, donor cells are harvested from the femur and tibia of genetically inbred mice and introduced into lethally irradiated syngeneic or allogeneic mice to reconstitute ablated hematopoietic compartments. In order to force reconstitution of myeloid cells from hematopoietic donors, myeloablative conditioning is employed. This can be done via TBI or via chemotherapy regimens. One note of caution however, is that different strains of mice have differing susceptibilities to TBI dosage. For instance, in our laboratory we routinely use 13 Gy split dose of TBI to myeloablate C57Bl/6 mice and only 10 Gy for the same purpose in Balb/c mice. Addition of splenic T cells along with the donor bone marrow inoculum can hasten reconstitution in syngeneic transplants  and 13 Gy TBI regimens induced greater than 50% reconstitution of AMs and spleen cells from donor stem cells by week 5 post-BMT   via intratracheal (i.t.) injection. Twenty-four hours post-infection, lungs and blood are collected to determine bacterial burden. Using this model, we found that syngeneic BMT mice are more susceptible to bacterial infections as they are unable to effectively clear P. aeruginosa   seem to have an overall pro-inflammatory effect on innate immune cells as they have been shown to promote bacterial phagocytosis and killing in both AMs and PMNs , however, suggest they may negatively regulate innate immune responses  receptors  and activation of the downstream cAMP targets: protein kinase A (PKA) and the exchange protein activated by cAMP (Epac-1) , AMs, and PMNs , another cytokine that is elevated in the lungs post-BMT and is produced by the AECs  in BMT AMs inhibits PGE2 signaling and restores phagocytosis of P. aeruginosa    , specifically Class A SRs   or COX-2-specific inhibitors  may prove beneficial to HSCT patients. More recently, the development of PF-044148948, the first specific EP2 antagonist developed by Pfizer (Wang, 2 signaling on AMs without shutting down all prostaglandin production or effecting other EP receptor signaling. Such a therapeutic may offer protection to HSCT patients suffering from antibiotic resistant bacterial infections. Finally, murine studies need to be confirmed in human HSCT AMs. If elevations in COX-2 and EP2 signaling as well as loss of SPA noted in murine studies can be verified in humans post-HSCT, the next step will be to design clinical trials to test therapies targeted at these transplant-induced alterations.Changes that characterize BMT AMs are diverse. Figure er Wang,  may offeThe 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.


Plasmodium falciparum and the human host, homologous and conserved interactions were inferred from various sources of interaction data. Such candidate interactions were assessed by applying a machine learning approach and further filtered according to expression and molecular characteristics, enabling involved proteins to indeed interact. The analysis of predicted interactions indicated that parasite proteins predominantly target central proteins to take control of a human host cell. Furthermore, parasite proteins utilized their protein repertoire in a combinatorial manner, providing a broad connection to host cellular processes. In particular, several prominent pathways of signaling and regulation proteins were predicted to interact with parasite chaperones. Such a result suggests an important role of remodeling proteins in the interaction interface between the human host and the parasite. Identification of such molecular strategies that allow the parasite to take control of the host has the potential to deepen our understanding of the parasite specific remodeling processes of the host cell and illuminate new avenues of disease intervention.To obtain candidates of interactions between proteins of the malaria parasite  Currently, little is known about large-scale protein interactions between cells, although large-scale maps are an important foundation for the understanding of the ways pathogens interact, invade and seize control of their human hosts. Recently, Uetz et al. released the first small map of computationally inferred physical protein interactions between the human host, the Kaposi-Sarcoma associated Herpesvirus and the Varicella-Zoster-Virus Plasmodium falciparumRecently, Vignali et al. released the first small map of experimentally determined protein interactions between the human host and the malaria parasite H. sapiens and the malaria parasite P. falciparum I inferred potentially conserved host-parasite interactions by utilizing orthologous protein groups. Furthermore, experimentally determined host-parasite interactions were used to generate potential interaction candidates by searching for organism-specific homologous proteins. To mitigate the potential influence of false-positive interactions, I applied a machine learning approach to assess the quality of the predicted interactions. Subsequently, predicted interactions between the human host and the parasite were filtered, accounting for parasite protein specific characteristics that are conducive to potential host-parasite interactions. In addition, I demanded that both parasite and human proteins were co-expressed in the corresponding parasitic cell cycle stages and human tissues/cells.Based on large-scale sets of species-specific protein interactions in P. falciparum and the human host. In comparison, the separate and combined sets of interactions shared similar characteristics. In particular, the pathogen seemed to utilize its protein repertoire in a combinatorial way by predominately targeting hub proteins. Such a strategy probably allows the parasite to take control of the human host cell, effectively reaching into signaling and other cellular functions of the host cell. Several prominent pathways of signaling and regulation proteins were predicted to interact with parasite chaperones, suggesting an important role of such proteins in the interaction interface between the human host and the parasite.The combination of predicted interactions with experimentally determined and structurally inferred interactions allowed for a large set of potential interactions between proteins of the malaria parasite Exploring a sequence orthology/homology based approach to determine protein-protein interactions between human host and parasite proteins, a flowchart of the procedure is shown in H. sapiens using large-scale high-throughput screens P. falciparum was utilized as well. Compiling 2,664 orthologous pairs of human and parasite proteins from the InParanoid database −6. Combining all sources, the final set was composed of 106,317 candidate interactions between 2,096 parasite and 8,650 human proteins.I assembled a network of 93,178 interactions between proteins of While sequence searches provided a large sample of candidates, such interactions most certainly contained a considerable amount of false positives. Assessing the quality of interactions I used the random forest algorithm rd of randomly picked training data. Subsequently, the corresponding true positive and false positive rates of the classification results were determined using the remaining ⅓rd of data. Repeating such random trials 1,000 times dot clouds in Using these sets as positive and negative training sets the random forest algorithm was applied to check the quality of candidate interactions. In particular, the algorithm provided the fraction of decision trees that reported a pair of proteins as interacting. Assuming different fractions as classification thresholds a ROC curve in Assessing the quality of candidate interactions with the random forest approach 12,406 interactions between 1,007 parasite and 3,614 human proteins were finally obtained.Putatively, only a subset of involved parasite proteins carried molecular characteristics that allowed interactions with the human host, prompting me to compile a list of 1,302 parasite proteins with such molecular features . FilteriIn addition, host-parasite interactions also were more likely to appear between human proteins that were expressed in liver tissue In the last step, the set of predicted interactions was combined with external data, such as experimentally determined and structurally inferred interactions, providing a total of 3,322 interactions between 388 parasite and 1,886 human proteins .P. falciparum and the human host the number of parasite proteins that targeted a host protein was calculated. vice versa, suggesting that many proteins were targeted by the parasite in a combinatorial way. External data such as experimentally determined and structurally inferred interactions and their combination with predicted interactions confirmed our initial observation.Utilizing predicted interactions between proteins of −4). Such a result indicates that the integrity of the predicted host-parasite interaction network was the result of a significantly non-random process.Considering topological integrity, the bipartite network of predicted interactions between parasite and host proteins was composed of 18 disconnected subnetworks. Notably, the largest subnetwork contained the vast majority of parasite and host proteins. By comparison, a bipartite network of external interactions was composed of 106 subnetworks, while a combined network broke into 62 components. If the choice of targeted host proteins were a random process a randomly assembled bipartite network would break into many more disconnected parts. In a null-model, parasite proteins were connected to the same number of randomly picked human proteins that were expressed in the erythrocyte and liver. Repeating these steps 10,000 times randomized networks significantly broke into a larger number of components than observed . Such observations strongly indicated that targeted human proteins effectively secured the parasite's reach into a breadth of signaling activities without inundating any particular one.As a further hypothesis, a parasite may effectively mediate the infection while ensuring variety given the tendency to target signaling pathway hubs. As a measure of diversity, the pathway participation coefficient (PPC) was defined: if a given protein predominantly interacted with partners that were members of the same pathway, PPC tends toward 1, while the opposite holds if the interaction partners of the considered protein were distributed among many different pathways. −30). Ward clustering of the matrix indicated a group of signaling pathways that were linked to 4 parasitic chaperone proteins  the best split from those variables is finally picked, and (iii) new data is predicted by aggregating the predictions of N trees.Random Forests is an ensemble learning method N = 343 triplet combinations and ⅔rd of all protein pairs were sampled. Since each interaction therefore ends up in the remaining ⅓rd several times (i.e. out-of-bag examples), the random forest algorithm reports the fraction of times the pair of proteins was classified as interacting. Specifically, an interaction was found if more than half of the decision trees voted that way (i.e. fraction f>0.5).For each of 1,000 decision trees TP), true negative (TN), false positive (FP) and false negative hits (FN). Furthermore, true positive and false negative rates were defined as Using out-of-bag examples as a cross-validation set a ROC curve was constructed by defining true positive Click here for additional data file.Table S2Negative training set including 1,136 interactions.(XLS)Click here for additional data file.Table S31,302 annotated parasite proteins that carry molecular characteristics, allowing them to interact with the human host.(XLS)Click here for additional data file.Table S4Combined set of 3,322 interactions including predicted (PRE) and external (EXT) interaction data.(XLS)Click here for additional data file.Table S5100 most enriched GO biological processes of host proteins that interact with parasite proteins in the combined set of host-parasite interactions.(XLS)Click here for additional data file.


Picoides arcticus) are emblematic of the role these disturbances play in creating wildlife habitat, since they are strongly associated with recently-killed forests. However, management practices aimed at reducing the economic impact of natural disturbances can result in habitat loss for this species. Although black-backed woodpeckers occupy habitats created by wildfire, prescribed fire, and mountain pine beetle infestations, the relative value of these habitats remains unknown. We studied habitat-specific adult and juvenile survival probabilities and reproductive rates between April 2008 and August 2012 in the Black Hills, South Dakota. We estimated habitat-specific adult and juvenile survival probability with Bayesian multi-state models and habitat-specific reproductive success with Bayesian nest survival models. We calculated asymptotic population growth rates from estimated demographic rates with matrix projection models. Adult and juvenile survival and nest success were highest in habitat created by summer wildfire, intermediate in MPB infestations, and lowest in habitat created by fall prescribed fire. Mean posterior distributions of population growth rates indicated growing populations in habitat created by summer wildfire and declining populations in fall prescribed fire and mountain pine beetle infestations. Our finding that population growth rates were positive only in habitat created by summer wildfire underscores the need to maintain early post-wildfire habitat across the landscape. The lower growth rates in fall prescribed fire and MPB infestations may be attributed to differences in predator communities and food resources relative to summer wildfire.Wildfire and mountain pine beetle infestations are naturally occurring disturbances in western North American forests. Black-backed woodpeckers ( Dentroctonus ponderosae, hereafter MPB) infestations are disturbances of particular interest to managers because of their widespread occurrence and economic impacts. Wildfires burned an average of 2.7 million ha annually between 2001 and 2011 Western North American forests are shaped by natural disturbances. From small-scale canopy gaps to stand-replacing fires, disturbances are an important source of heterogeneity and species diversity Picoides arcticus) are emblematic of the role these natural disturbances play in creating wildlife habitat. Black-backed woodpeckers are associated with habitat created by wildfire Black-backed woodpeckers  forests in the Cascade Mountains of Oregon Pinus ponderosa) forests in the Black Hills of South Dakota Pseudotsuga menziesii) forests in British Columbia Although typically associated with post-fire habitat, black-backed woodpeckers are also attracted to MPB infestations. Black-backed woodpeckers use MPB infestations in lodgepole pine , paper birch (Betula papyrifera), and white spruce (Picea glauca) This study occurred in the Black Hills, South Dakota at study sites representing habitat created by wildfire, prescribed fire, and MPB infestations . Study sWe used very high frequency radio-transmitters  to collect survival data from adult and juvenile black-backed woodpeckers. We initially targeted adult woodpeckers for capture by searching study sites for signs of woodpeckers. We then used mist nets, hoop nets, and netguns n = 25) were fitted with a 2.2 g transmitter n = 6). Nestlings weighed an average of 57 g (SD = 10 g) and transmitters weighed <5% of an average nestling’s mass We captured black-backed woodpecker nestlings at the nest cavity with a ‘chick-snagging’ device and by accessing the nest with a hole-saw We located nests by systematically searching study areas, by following birds to their nests, and opportunistically while collecting field data. We visited nests every 3–4 days during the 2009–2011 breeding seasons until either the nest failed or fledged young. Because of logistic constraints, nest visits were less regular during the 2008 and 2012 field seasons and the mean interval between nest visits was 4 days. We examined nest contents with a camera attached to a telescoping pole The majority of field work occurred on public land . We obtained permission to access Wind Cave National Park from the US National Park Service, permit number WICA-2010-SCI-0011. Two authors (MAR and CPL) were employees of the US Forest Service and Custer State Park, respectively. We therefore did not require specific permission to access the Black Hills National Forest or Custer State Park. We avoided private lands when possible, and obtained permission from landowners when necessary to access private land .All birds were individually marked and fitted with transmitters as quickly as possible to minimize stress. All research protocols were approved by the University of Missouri Animal Care and Use Committee, protocol numbers 4439 and 7205. Black-backed woodpeckers are protected under the Migratory Bird Act. Permission to capture and band black-backed woodpeckers was thus obtained from the US Department of the Interior, Federal Bird Banding Permit number 23574 and Federal Fish and Wildlife Permit number MB161645-0; and from the State of South Dakota Department of Game, Fish, and Parks, Free Scientific Collector’s Permit License Number 10.itd, is the probability woodpecker i is detected by the end of time step t. Survival probability, denoted φit, is the probability woodpecker i is alive at the end of time step t. We thus modeled a woodpecker’s state at time t as a multinomial random variable:Mt−1 is the row of the state transition matrix associated with a woodpecker’s state at time t–1.We estimated adult and juvenile survival probabilities with Bayesian multi-state mark recapture models i had an active transmitter during time step t. Juveniles that were color-banded but did not receive a transmitter were coded as having an inactive transmitter. We modeled monthly adult survival probability as a function of sex, season , the habitat occupied at the end of each time step , and number of years post-fire. We modeled bi-weekly juvenile survival probability as a function of habitat, the number of years post-fire, and the number of two-week time intervals since fledging. Details of covariate models and prior distributions are described in st April post-fledging, which corresponds with the beginning of the breeding season.We modeled adult and juvenile detection and survival probability during each time step as a function of covariates. We assumed a 1-month time step for adult detection and survival probability and a 2-week time step for juvenile detection and survival probability. We modeled adult and juvenile detection probability as a function of whether woodpecker Woodpeckers inhabiting established study sites were assigned the habitat category associated with that study site .Adult survival probability was greatest in habitat created by wildfire, intermediate in habitat created by MPB infestations, and lowest in habitat created by prescribed fire . For exaWe censored the detection history for 1 juvenile that died before fledging. After censoring, we estimated juvenile survival probabilities from detection histories of 72 woodpeckers, consisting of 119, 139, and 44 cumulative time steps  at risk of dying in habitat created by wildfire, MPB infestations, and prescribed fire, respectively. The average period between first and last detection of individual juveniles was 10 weeks (range = 2 to 42 weeks).Patterns of habitat-specific juvenile survival probability closely tracked patterns of habitat-specific adult survival probability. The mean probability of juvenile black-backed woodpeckers surviving to the adult stage class  and nearly identical between habitat created by wildfire  and MPB infestations  were positive only in habitat created by wildfire. For example, Mean posterior distributions of population growth rates . Second, the prescribed fire study sites tended to be smaller in area, burn at lower severity, and thus kill fewer trees relative to the wildfire study sites, which may result in different predator communities between the two disturbance types Habitats created by wildfire and prescribed fire differed at our study sites in two primary ways. First, managers treated sites with prescribed fire in September and October, while wildfire sites burned during June and July. This difference in timing may affect post-fire arthropod communities. For example, some species of wood-boring beetles can detect compounds emitted from burning wood Our sensitivity analysis provides insight into how these potential differences may affect population growth rates. In all habitats, population growth rates were most sensitive to changes in adult and juvenile survival. If the timing of prescribed fire affects wood-boring beetle abundance, this may affect the food resources available to black-backed woodpeckers, which may in turn impact survival rates. This may particularly affect juvenile survival, since fledglings rely on provisioning from adults for several weeks post-fledging. Survival rates may also be affected by potential differences in predator communities between habitats. For example, Northern Goshawks, a known predator of black-backed woodpeckers in the Black Hills We were surprised that mean population growth rates were negative in habitat created by MPB infestations, since woodpeckers readily occupy and successfully breed in such habitat Habitat created by MPB infestations likely harbors some value to black-backed woodpeckers, even if mean population growth rates in this habitat were negative. Indeed, 95% CIs of estimated growth rates overlapped 1, suggesting the potential for positive population growth in this habitat during some years. It is likely that mean population growth rates in MPB infestations are intermediate between early post-wildfire habitat and undisturbed forest, although additional research is needed to understand demographic rates of black-backed woodpeckers in undisturbed forests. Additionally, adult, juvenile, and nest survival all declined as burned forests aged and mean population growth rates in 4-year post-wildfire forests were similar to MPB infestations . Mountaith century, including the current infestation The differences in population growth rates between habitat created by wildfire and MPB infestations likely reflects historic disturbance patterns in the Black Hills. There were 7 discrete MPB outbreaks in the Black Hills during the 20We do not wish to completely discard the utility of either prescribed fire or MPB infestations as important disturbance agents for black-backed woodpeckers. Large, severe wildfires are not likely to gain widespread acceptance on public lands, and prescribed fire may be the only way to predictably introduce fire. Prescribed fire is a flexible management tool that can be applied in a variety of ways. For example, managers can vary factors including timing, severity, and extent. Further research is needed to determine the mechanisms leading to negative population growth of black-backed woodpeckers in habitat created by prescribed fire so forests can be treated in an appropriate manner. In particular, further research is needed to understand how the timing of prescribed fire may impact post-fire wood-boring beetle abundance, a primary food resource for black-backed woodpeckers Although our study evaluates demographic rates of black-backed woodpeckers over a range of disturbed forest conditions, black-backed woodpeckers are also known to use undisturbed forest across their range Our study is the first to evaluate the demographic response of black-backed woodpeckers to a range of disturbance conditions. This and other studies are necessary to gain a better understanding of the importance of wildfire and other forest disturbances in maintaining viable black-backed woodpecker populations. Our results indicate this species is dependent on early post-wildfire habitat in the Black Hills, South Dakota, underscoring the importance of ensuring recently burned forest is present across the landscape.Figure S1Home ranges of black-backed woodpeckers relative to the 4-Mile wildfire in Custer State Park, SD. The gray shaded polygon represents the extent of the 4-Mile wildfire, individual points represent coordinates of black-backed woodpecker locations obtained via radio-telemetry, and dashed lines represent 95% probability contours of black-backed woodpecker home ranges estimated using kernel density methods (TIF)Click here for additional data file.Figure S2Home ranges of black-backed woodpeckers relative to the East Slate Creek mountain pine beetle infestation. The green shaded polygons represent <1 year old mountain pine beetle (MPB) infestations as of autumn 2009 (TIF)Click here for additional data file.Figure S3Results from life stage simulation analysis evaluating sensitivity of black-backed woodpecker asymptotic population growth rates. Each point represents an estimate of asymptotic population growth rates (λ) corresponding to a random realization from the posterior distribution of A) annual adult survival probability, B) the probability a juvenile survives to an adult, C) the probability a nest successfully fledges at least 1 young, and D) the expected number of females fledged per successful nest. The coefficient of determiniation (r2) is calculated by regressing λ against the corresponding demographic rate (TIFF)Click here for additional data file.Table S1Summary of posterior distributions of parameters included in the adult survival model.(PDF)Click here for additional data file.Table S2Summary of posterior distributions of parameters included in the juvenile survival.(PDF)Click here for additional data file.Table S3Summary of posterior distributions of parameters included in the nest survival model.(PDF)Click here for additional data file.Table S4Summary of posterior distributions of parameters included in the number fledged model.(PDF)Click here for additional data file.Appendix S1Statistical methods for modeling demographic rates of black-backed woodpeckers.(PDF)Click here for additional data file.


Then, the weighted miRNA-target networks were constructed for different TCM syndromes using predicted miRNA targets. Subsequently, the prioritized target genes of upexpression network of TCM syndromes were analyzed using DAVID online analysis. The results showed that there are distinctly different hierarchical cluster and network structure of TCM syndromes in HCC, but the excessive-deficient combination syndrome is extrinsically close to deficient syndrome. GO and pathway analysis revealed that the molecular mechanisms of excessive-deficient and deficient syndromes of HCC are more complex than excessive syndrome. Furthermore, although excessive-deficient and deficient syndromes have similar complex mechanisms, excessive-deficient syndrome is more involved than deficient syndrome in development of cancer process. This study suggested that miRNAs might be important mediators involved in the changing process from excessive to deficient syndromes and could be potential molecular markers for the diagnosis of TCM syndromes in HCC.Traditional Chinese medicine (TCM) treatment is regarded as a safe and effective method for many diseases. In this study, the characteristics among excessive, excessive-deficient, and deficient syndromes of Hepatocellular carcinoma (HCC) were studied using miRNA array data. We first calculated the differentially expressed miRNAs based on random module  Hepatocellular carcinoma (HCC) is one of the most common devastating cancer types, ranking sixth in incidence, and the third leading cause of cancer related deaths worldwide , 2. The Under these circumstances, many failing patients seek to get help from complementary and alternative medicine, especially traditional Chinese medicine (TCM). TCM is an ancient Chinese medicine that evolved through at least three thousand years of uninterrupted clinical practice. The TCM treatment usually uses a traditional diagnosis method to classify the TCM syndromes, which based on clinical symptoms and signs, followed by the use of individualized treatment , 6. For MicroRNAs (miRNAs) are endogenous, noncoding, single-stranded small RNA molecules of approximately 22 nucleotides, which function as negative regulators that involve posttranscriptional gene expression through binding to the 3′-untranslated regions (3′-UTRs) of target mRNAs and consequently lead to mRNA cleavage or translational repression –11. AlthIn this work, we hypothesized that miRNAs expression levels and their target gene expression networks are the important factors for the TCM syndrome classification and the changes from excessive to deficient syndromes in HCC patients. We thus focused on the comparative analysis of the differences and similarities in the three TCM syndromes including liver-gallbladder dampness heat syndrome (LGDHS), liver depression and spleen deficiency syndrome (LDSDS), and liver-kidney yin deficiency syndrome (LKYDS). The aim is to demonstrate the change process of molecular mechanism from excessive syndrome to deficient syndrome at a network level by an integrative and comparative analysis of weighted miRNA-target network in HCC patients.In this work, clinical serum of 9 HCC patients and 7 healthy donors  were collected, whose were come from Shanghai Longhua Hospital. Then, these serums were subjected to miRNA microarray analysis. The diagnostic criteria of western medicine for HCC followed the guidelines defined by the Chinese Society of Hepatology and Chinese Society of Infectious Diseases in 2005 . The TCMThe differentiation of TCM syndromes in HCC patients was shown in μL.All serum samples were from the peripheral venous blood of HCC patients and healthy donors, which were immediately frozen in liquid nitrogen and then stored at −80°C. The RNAs in serum were extracted using a miRVana PARIS kit  according to the manufacturer's protocol and using the RNase-free DNase  to eliminate DNA contamination. The concentration of RNAs isolated from serum ranged from 1.5 to 12 ng/The profiles of serum miRNAs of 9 HCC patients and 7 normal controls were generated using Agilent Human miRNA microarray V3 ; 60 ng of RNA was labeled and hybridized for each array. Hybridization signals were detected with the Agilent microarray scanner; the data were extracted using Feature Extraction V10.7 . All raw data were transformed into log 2 scale, and then, the expression levels were normalized by having zero mean and unit sample variance.t-test and the fold-change >1.5 and P < 0.05 were considered significant. Heat-map analysis and hierarchical cluster analysis of expression data were performed using Cluster 3.0 and TreeView programs . Class prediction was performled using a statistical algorithm of the support vector machine (SVM) incorporating miRNA differentially expressed at a univariate parametric significance level of P = 0.01. The prediction rate was estimated via 10 fold and 10 times cross-validation and the bootstrap method for small sample data.In order to evaluate the diversity of three TCM syndromes in HCC, we compared the miRNAs expressions of LGDHS, LDSDS, and LKYDS to normal, respectively. The relative miRNA expression levels were further normalized utilizing the median over the all patients, which make the each patient have a median log ration of 0 in normalized expression levels. The weighted differences miRNAs between TCM syndromes were calculated using the random variance model Validated miRNA target genes were selected based on TarBase 6.0, which hosts the largest collection of manually curate experimentally validated miRNA-gene interactions . FurtherP < 0.05) expression difference between normal, LGDHS, LDSDS, and LKYDS samples were analyzed for pathways involving these genes using DAVID online analysis [P values were corrected for false discovery rate (FDR). Gene sets containing less than 5 genes overlapping were removed from the DAVID analysis. In our analysis, GO terms and pathways with an FDR-adjusted P value of less than 0.05 were retained.Of the inferred miRNA target genes, those showing a significant , while target genes were weighted based on degree distributions between consecutive groups, and thus we obtained a node-weighted miRNA-target interaction network for each stage. In order to validate the veracity of above network, rank all nodes (miRNAs and target genes) of network according to their weights and test the similarity between them , 22; thet-test of R package, to search whether there are some significantly differential expressed miRNAs among the consecutive stages form excessive syndromes to deficient syndromes. 35 miRNAs in LGDHS/normal, 61 miRNAs in LDSDS/normal, and 71 miRNAs in LKYDS/normal differentially expressed. Three TCM syndromes of HCC were classified using a supervised learning algorithm (binary tree classification), and SVM act as prediction method. As shown in The expressions of miRNAs were calculated and analyzed with random module Hierarchical cluster analysis revealed that the expression profiles of the differential miRNAs from each TCM syndrome were roughly classified, respectively. The consecutive heatmaps of differentially expressed miRNAs were shown in P < 0.05) in each prediction program. Following the differential expressed miRNAs among LGDHS, LDSDS, and LKYDS in HCC, miRNA-target network for each syndrome was reconstructed based on predicted data. The global profiles of networks were shown in As a gene regulator, a given miRNA usually has multiple different mRNA targets, and multiple miRNAs might target one gene . In thisTo reveal the details of the regulatory core of network, the simplified network was reconstructed through the selected hub nodes. In this work, we defined a hub node that has more than 5 interactions in those TCM syndrome-specific networks, which represent these hub nodes might have highly effects for the topological structure of network. The reconstructed networks were shown in Because upexpression miRNAs might play roles that are more important in biological process, we divided the up-expression miRNA-target networks from the holistic networks for each TCM syndrome. P values were corrected for false discovery rate (FDR). Gene sets containing greater than 5 genes overlapping were retained from the DAVID analysis. Analysis results with an FDR-adjusted P value of less than 0.05 were retained.Because miRNA general inhibit translation or induce mRNA degradation by binding to the 3′-UTRs of target mRNAs ; here, wP value and FDR less than 0.05. The distribution of overlapping GO terms was shown in P value of each GO term is different. For instance, the P values of GO: 0003677 (DNA binding), GO: 0031981 (nuclear lumen), GO: 0044451 (nucleoplasm part), GO: 0048522 (positive regulation of cellular process) and GO: 0070013 (intracellular organelle lumen) in LKYDS are clearly less than LGDHS or LDSDS. These results implicated that these GO terms are more stringently associated with deficient syndrome (LKYDS) and could deregulate the core cellular functions.The representative GO and pathways terms of target genes in each TCM syndrome were shown in The pathway terms (KEGG and BIOCARTA) of target genes were also calculated using DAVID analysis. As shown in The detail of each pathway of three TCM syndromes was represented in In this study, based on miRNA microarray date of HCC patients and normal controls, we obtained evidence that the miRNA expression profiles of LGDHS, LDSDS and LKYDS are different. The classification results show that the Deficient syndrome (LKYDS) first cluster to Excessive-deficient syndrome (LDSDS), then as a parallel branch cluster to Excessive syndrome (LGDHS). Furthermore, the lower SVM score between LDSDS and LKYDS also suggested that the excessive-deficient syndrome is extrinsically close to deficient syndrome in HCC patients. The topological structure showed that the hub nodes (miRNAs or target genes) of miRNA-target network are much stronger determinants of the realized gene expression profiles, whereas the periphery nodes that should be regulated are not regulating. Obviously, the different topological profiles of networks also involved in the molecular mechanisms are different form Excessive to Deficient syndromes in HCC. GO and pathway analysis of target genes in up-expression networks revealed that the excessive-deficient and deficient syndromes of HCC are more complexity than excessive syndrome. Furthermore, although excessive-deficient and deficient syndromes have similar complex mechanism, excessive-deficient syndrome might have more dangers than deficient syndrome involved in development of cancer process.


The airways do not normally manifest nociceptive pain from a stimulus but the only consistent response that capsaicin and lung inflammation provoke in healthy human airways is cough. TRPA1, another excitatory ion channel, has been referred to as the "irritant receptor" and its activation also induces cough. For both types of cough, the motor responses are identical and via coordinated, precisely-timed and sequential respiratory events orchestrated by complex neuromuscular networking of the diaphragm, chest and abdominal respiratory muscles, the glottis and parts of the brain.This review dissects the complex human cough reflex and suggests hypotheses about the evolutionary basis for the reflex. A mechanosensory-induced cough reflex conveys through branches of myelinated Aδ nerve fibers is not chemically reactive ; possibly, its evolution is to prevent the harmful effects of aspiration of gastric or particulate contents into the lungs. This became necessary as the larynx moves closer to the opening of the esophagus as human ancestors adapt phonation over olfaction beginning less than 10 million years ago. The second type of cough reflex, a chemosensory type, is carried by unmyelinated C fibers. Supposedly, its origin dates back when prehistoric humans began living in close proximity to each other and were at risk for infectious respiratory diseases or irritant-induced lung injury. The mechanism for the latter type of cough is analogous to induced pain after tissue injury; and, it is controlled by the identical transient receptor potential vanilloid cation channel (TRPV Persistent cough is one of the most common medical complaints that impacts the quality of life and is responsible for a significant proportion of annual ambulatory medical visits and medical expenses in the United States . InvolveWhen you cough there are thousands of unseen muscles that suddenly spring into action. It's like watching that fat guy catch a cannonball in his stomach in slow motion" .Considering a casual connotation, coughing is a reflex-evoked modification of normal breathing patterns . More exAccordingly, I am attending a dinner with my wife and observe her walking across the room towards me. As she does so, she aspirates a sliver of ice from a glass of beverage she is drinking. My wife stops walking and her eye brows arch. She informs me later that at that instance she experienced the sensation of an urge to cough. She deftly places a clenched fist to her mouth as her face reddens; her eyes water, narrow and then tighten. Her chin lifts to some extent as her head rears back; perspiration appears on her upper lip. Her stance widens. Her back bends slightly backwards. Her chest expands as she takes in a breath. She holds her breath momentarily only to quickly open her mouth again. Now, her face is cerulean-colored as she forcibly emits a staccato-like exhalation. Then, at the very end of her explosive exhalation, she daintily wipes a small speckle of ice from her lower lip using a pink-colored lace handkerchief that she inconspicuously hides in the cleavage of her dress. As she looks across the room at me, she smiles and nonchalantly lifts both her shoulders and spreads her arms out with the palms facing upwards as if to denote a sense of embarrassment.For my wife, it is reasonable to believe that instigation of a cough response is due to stimulation of a mechanically-sensitive 'true cough receptor' that is provoked by the sliver of ice. The premise for the presence of a 'true cough receptor' is explored by Canning et al using the anesthetized guinea pig model, which retains a blunted cough response following noxious stimuli ,6. MechaPurportedly, the vagal afferent neurons of the true cough receptor are unlike the rapidly adapting receptors (RARs), slowly adapting receptors (SARs) or C-fibers; also, this receptor does not express transient receptor potential vanilloid (TRPV1) and is not sensitive to capsaicin. In contrast to the anesthetized guinea pig model, mechanically-induced coughing in conscious guinea pigs generates impulses carried by low threshold, mechanically sensitive, rapidly adapting receptors (RARs) that travel through myelinated Aδ fibers at conduction velocities of between 4 and 18 m/s -17. ThesTracheal and laryngeal receptors come in to play as an important defensive role against acid aspiration . The 'tr2, citric acid, hypertonic saline solution, SO2 and lung inflammation  . T. T108]. piration ,108. TheHomo species as they adapt speech over olfaction. The genes regulating brain size and behavior exhibit higher rates of protein evolution in the lineage leading from ancestral primates to humans [ASPM) may have been the evolutionary target for the initial expansion of the hominid cerebral cortex; and, changes regarding human speech are likely accelerated after the FOXP2 regulatory gene reaches its modern normal variant around 100,000 years ago [The growth in the size of the human cortex permits better control of voluntarily speech and song production  as opposed to the limited voluntary control over vocalizations displayed by non-human primates ,112-114.ears ago -123.Homo species fashion stronger social connections. There emerges an imperative need to defend against distal lung inflammation or damage as Homo chronospecies move to more enclosed environs where there is a greater possibility of contracting a contagious respiratory tract or parasitic infections and/or being exposed to gaseous-particulate irritant emanations [Homo species, possibly beginning 400,000 years, are supposedly within caves requiring fire for warmth and for cooking of food [The second type of human cough reflex is adapted as the capacities for speech and cognition evolve and anations ,125. Pulanations ,124-127. of food . Fire re of food . BiomassTeleologically, the lung C-fiber neural responses may permit a broader defensive rejoinder than does just coughing, a warning sign like pain. Possibly, cough associated with respiratory infections is a coevolutionary strategy by primitive viruses coexisting with human predecessors; induction of coughing increases viral contagiousness to enhance viral spread and survival ,130. TheHomo chronospecies move to more enclosed environs where there is a greater possibility of contracting a contagious respiratory tract or parasitic infections and/or exposure to gaseous-particulate irritant emanations. Homo species fashion stronger social connections with the introduction of speech (over olfaction) and the enlargement of the cortical brain size. Homo species (and perhaps an earlier hominid) achieve voluntarily cough initiation and suppression; control of the forced exhalation and the intensity of the cough response; the facility to initiate repeated coughing; a heeding to the integration of psychosocial factors into the cough response; exquisite control of laryngeal muscles actions; a capacity to respond to a variety of cough stimuli; and the perception of airways irritation causing an "urge to cough".A mechanosensory reflex cough, receptive to mechanical and acid stimulations, possibly operates via a mechanism involving the so-called "true cough receptor"; or possibly, there is another mechanism employing other slowly conducting nerve fibers. Pertinent evolutionary adaptations shaping the first type of human reflex cough response comprise modifications of the supralaryngeal tract and descent of the hyoid bone and movement of the larynx to a closer location to the esophageal opening, which increases the risk for aspiration. A second, chemosensory-type reflex cough, originating in some more ancient mammal, involves C-fiber afferent nerves linked to lung inflammation and chemical agents . The need for defending against distal lung inflammation or damage emerges as 1TRPV: Transient Receptor Potential Cation Channel Subfamily V, Member 1; 1TRPA: Transient Receptor Potential Cation Channel, Subfamily A, Member 1; Aδ Nerve Fibers: A Delta Fibers (Afferent Fibers); C Nerve Fibers: Unmyelinated C fibers (Afferent Fibers); SARs: Slowly adapting Fibers; RARs: Rapidly Adapting Receptors; ASIC:Acid-Sensing Ion Channel-Type Receptor; 2PGE: Prostaglandin E2; BOLD:Blood Oxygen Level-Dependent; 2FOXP: Forkhead Box Protein P2The author declares that they have no competing interests.Dr. Stuart M Brooks is currently Adjunct Professor in the College of Public Health, University of South Florida.


Little is known about the changes of brain structural and functional connectivity networks underlying the pathophysiology in migraine. We aimed to investigate how the cortical network reorganization is altered by frequent cortical overstimulation associated with migraine.Gray matter volumes and resting-state functional magnetic resonance imaging signal correlations were employed to construct structural and functional networks between brain regions in 43 female patients with migraine (PM) and 43 gender-matched healthy controls (HC) by using graph theory-based approaches. Compared with the HC group, the patients showed abnormal global topology in both structural and functional networks, characterized by higher mean clustering coefficients without significant change in the shortest absolute path length, which indicated that the PM lost optimal topological organization in their cortical networks. Brain hubs related to pain-processing revealed abnormal nodal centrality in both structural and functional networks, including the precentral gyrus, orbital part of the inferior frontal gyrus, parahippocampal gyrus, anterior cingulate gyrus, thalamus, temporal pole of the middle temporal gyrus and the inferior parietal gyrus. Negative correlations were found between migraine duration and regions with abnormal centrality. Furthermore, the dysfunctional connections in patients' cortical networks formed into a connected component and three dysregulated modules were identified involving pain-related information processing and motion-processing visual networks.Our results may reflect brain alteration dynamics resulting from migraine and suggest that long-term and high-frequency headache attacks may cause both structural and functional connectivity network reorganization. The disrupted information exchange between brain areas in migraine may be reshaped into a hierarchical modular structure progressively. Migraine is an idiopathic headache disorder that has been an important healthcare and social problem for its great influence on the quality of life, accompanied by severe headaches, nausea and light sensitivity Due to frequent migraine-related nociceptive input, migraine has had serious secondary effects upon the central nervous system (CNS) To be able to fully understand structural/functional connectivity patterns, a comprehensive map of connection patterns of the human brain is needed All research procedures were approved by the West China Hospital Subcommittee on Human Studies and were conducted in accordance with the Declaration of Helsinki. All participants in our study gave written informed consent.Forty-three right-handed migraine patients , 14.5±6.8 years (mean migraine duration± SD)) were recruited . The migFor both groups, exclusion criteria were: 1) macroscopic brain T2-visible lesions on MRI scans; 2) existence of a neurological disease; 3) pregnancy or menstrual period; 4) use of prescription medications within the last month; 5) alcohol, nicotine or drug abuse; and 6) claustrophobia. All subjects gave written, informed consent after the experimental procedures had been fully explained.2, data matrix = 64×64). For each subject, a total of 205 volumes were acquired, resulting in a total scan time of 410 s. Subjects were instructed to rest with their eyes closed, not to think about anything in particular, and not to fall asleep. After the scan, the subjects were asked whether they remained awake during the whole procedure.All subjects underwent a resting-state functional MRI scan using a 3T magnetic resonance system  with an 8-channel phased array head coil. Prior to the functional run, a high-resolution structural image for each subject was acquired using three-dimensional MRI sequences using an axial Fast Spoiled Gradient Recalled sequence (3D-FSGPR) . The functional images were obtained with an EPI . The average gray matter volumes were estimated. Specifically, the structural images were first corrected for non-uniformity artifacts; second, by using nonlinear normalization, the corrected images were registered to an asymmetrical T1-weighted template; third, the gray matter, white matter, and cerebrospinal fluid were obtained from then normalized images; and fourth, the resulting gray matter images were smoothed by a 4 mm isotropic Gaussian kernel.The structural images were processed using voxel-based morphometry (VBM) with Statistical Parametric Mapping-5 (SPM5)  template images were used to parcellate the entire cerebral cortex into 90 anatomical regions of interests (ROIs) [33]. TheFor brain structural networks, the average gray matter volumes within each ROI (n = 90) were calculated. We measured the correlations based on the ROIs' averaged gray matter volumes For brain functional networks, we measured the regions' mutual association, expressing their functional coupling to define the network connection The structural/functional connections of the cortical networks were defined if the correlation coefficient in the above structural/functional connection matrix between the two nodes achieved a correlation threshold. In the current study, the sparsity value used a network threshold which was defined as the total number of edges in a network divided by the maximum possible number of edges The clustering coefficient of a node 0< Ci <1 is a ratio that defines the proportion of possible connections that actually exist between the nearest neighbors of a node where N is the total number of nodes in the network, The mean clustering coefficient of network C is the average over each node's clustering coefficient:The minimum path length where Corresponding parameters for a random graph of C and L with the same number of nodes were also calculated, as denoted by The betweenness centrality of the brain regions was defined as the number of shortest paths between any two nodes in the network that pass through that particular node j to node k, and j to node k that passes through node i.where i and j, i belongs to group 1 and i belongs to group 2. The expected number of edges linked at random between nodes i and j is Modularity Q of the brain cortical networks is defined as To estimate whether there existed significant group differences in the functional connections, a recently developed network-based statistic (NBS) was used in the current study p<0.01, corrected) and normalized clustering coefficient p<0.01, corrected) were significantly higher in PM structural networks on a whole sparsity range. No significant differences were found in the shortest absolute path length L and p>0.05). Moreover, similar results were found in patients' functional networks. The comparisons revealed significantly increased clustering coefficients C and p<0.01, corrected), but they had an unaltered shortest absolute path length as compared with HC. Furthermore, the mean clustering coefficient C was positively correlated with the duration of migraine in the patients' functional networks . No significant correlation was found in the shortest absolute path length L , including the PreCG, dorsolateral part of the superior frontal gyrus (SFGdor), PHG, ACG, THA, amygdala (AMYG), TPOmid and IPL. Furthermore, some of these regions showed a significant correlation with migraine duration . We focused on the largest connected component, and these abnormal connections were widely distributed in the patients' structural networks including connections between different lobes, mainly in the ACC, THA, hippocampus (HIP), caudate (CAU), putamen (PUT), pallidum (PAL), orbital part of the prefrontal cortex, parietal lobule, temporal lobes, and occipital cortex. All of these connections revealed increased values in patients compared with HC . The functional network of patients was more dysregulated as compared with their structural networks, and connections showed significant increases in interregional correlations   .Modularity is defined as a set of nodes the have many intra-modular connections but sparser inter-modular connections, indicating a decomposability of the system into smaller subsystems In this study, we presented graph theoretical analyses of topological properties of whole-brain networks by examining the structural and functional connectivity network disorganization in migraine sufferers as compared with HC. Exploring the similarity of functional and structural network changes, we found that migraine disrupts the topological organization of cortical networks, and the vital brain hubs related to pain-processing revealed abnormal nodal centrality in both structural and functional networks; brain alteration occurring in patients with migraine was not limited to the local abnormal CNS but to a disruption in the topological organization of intrinsic whole-brain networks. Furthermore, disrupted information exchange between brain areas was reorganized into a hierarchical modular structure. Taken together, our results indicated that long-term and high-frequency headache attacks may lead to pathological cortical network reorganization in PM, reflecting abnormal brain dynamics due to the effects of brain disease. It has profound implications for our understanding of topological organization of complex brain networks in migraine.In our findings, both structural and functional connectivity networks of the PM showed significantly increased clustering coefficients over a wide range of sparsity values as compared with HC. Given that the clustering coefficient is an index of local structure, such results could be attributed to the increased degree that more brain regions in patients tend to cluster together, involving networks comprised of pain processing and the visual cortex . These tIn our results, compared with the matching HC, several abnormal interregional correlations were found in PM cortical networks and were formed into a large connected network, which refers to several brain circuits . NotablyPrior studies have noted the importance of modular structure in the biological networks which may arise from natural selection pressure or evolutionary constraint for adaptation to environmental demands There are several issues that need to be further addressed in the present study. First, in our network analysis, we employed AAL template images and had 90 ROIs covering the entire cerebral cortex which were used in several published studies In summary, by exploring the topological properties of the structural and functional connectivity networks in patients with migraine, the patients showed a dysregulated brain organization in their cortical networks. This migraine-affected brain alteration had a skewed balance between structural and functional connectivity networks in patients. More abnormal brain connections were found in migraine sufferers; furthermore, disrupted information exchange between brain regions was formed into a hierarchical modular structure. Our results provide valuable insights into the understanding of brain network reorganization that could be attributed to developmental aberration and the underlying pathophysiology resulting from migraine. In our future longitudinal study, direct evidence will be provided to prove that migraine is a progressive disease and results in dysregulated brain network reorganization.


Carcinosarcoma is a rare biphasic neoplasia containing both malignant mesenchymal and epithelial elements. Bladder carcinosarcoma commonly presented as high-grade, advanced stage, and aggressive behavior with a poor prognosis. An 83-year-old male presented with painless gross hematuria to our hospital. Cystoscopy revealed massive nonpapillary bladder tumor on the right wall. The 91 g tumor could be completely removed with transurethral resection. Histology of the tumor was diagnosed as carcinosarcoma with no submucosal invasion composed of biphasic malignant epithelial and mesenchymal cells. Epithelial malignancy was urothelial cancer and mesenchymal one was chondrosarcoma and leiomyosarcoma. The specimens taken at the second-look TUR-Bt revealed that carcinoma in situ  but not sarcoma existed at the mucosa surrounding the previous tumor site. 80 mg of BCG instillation intravesically every week for six weeks was successfully administered to the patient. There is no tumor recurrence for 6 months after treatments. Carcinosarcoma, also called true malignant mixed tumor, is a rare biphasic neoplasia containing both malignant mesenchymal and epithelial elements. Only 79 cases of carcinosarcoma of bladder origin have been reported in the English literature in the year of 2007 . BladderThe mesenchymal element of carcinosarcoma lacks epithelial markers , 4 and pIn cases of bladder carcinosarcoma, evidence supporting a monoclonal origin for the epithelial and mesenchymal components was revealed using loss of heterozygosity studies with microsatellite markers , 7, whilAn 83-year-old male presented with painless gross hematuria to our hospital.Cystoscopy revealed massive nonpapillary bladder tumor on the right wall. Pelvic CT scan showed a giant bladder tumor more than 10 cm in diameter , while tHistology of the tumor was diagnosed as carcinosarcoma composed of biphasic malignant epithelial and mesenchymal cells. Epithelial malignancy was urothelial cancer  and meseConsidering the noninvasive pathology of the tumor despite well-known aggressive behavior of carcinosarcoma, it was difficult to judge if more invasive treatments such as radical cystectomy or external beam radiation therapy + interventional arterial infusion of anticancer drug(s) are mandatory. Thus, second-look TUR-Bt was done 7 weeks following the first TUR-Bt. The specimens taken at the second-look TUR-Bt revealed that there was no remaining tumor cells at the site where the previous tumor was resected by the first TUR-Bt, while carcinoma in situ  but not sarcoma existed at the mucosa surrounding the previous tumor site (data not shown). Then, we forwarded to the intravesical BCG instillation therapy to the patient and 80 mg of BCG every week for six weeks was successfully administered. Pelvic, abdominal, and chest CT scans as well as cystoscopy and urine cytology have not shown tumor recurrence for 6 months after treatments.A case of bladder sarcomatoid carcinoma, which was resected by TUR-Bt and diagnosed as nonmuscle invasive (pT1 stage), was previously reported to have shown intravesical recurrence during followup, undergone partial and total cystectomy, and finally died 13 months after diagnosis . On the In general, more aggressive therapies than TUR-Bt alone are usually adopted as treatments for bladder carcinosarcoma due to invasiveness of the tumor. The pT stage of our case was fortunately pTa without submucosal infiltration if we simply apply the pathological staging system of bladder urothelial cancer and the second-look TUR-Bt revealed carcinoma in situ but not already invasive cancer/sarcoma, then we did not perform radical cystectomy or chemoradiotherapy in our case, but intravesical BCG instillation therapy trying to preserve bladder. It might have been fortunate that the tumor base was mainly composed of urothelial cancer but not sarcoma, as the latter may often be more invasive than the former. Of course, the patient naturally needs close followup for tumor local recurrence and metastasis. CT scans, cystoscopy, and urine cytology every three months may be necessary during the first year after treatment.


Therefore, we tested the hypothesis that CPAP restores the link between cardiac autonomic activity and delta sleep across the night.Continuous positive airway pressure (CPAP) treatment improves the risk of cardiovascular events in patients suffering from severe sleep apnea-hypopnea syndrome (SAHS) but its effect on the link between delta power band that is related to deep sleep and the relative cardiac vagal component of heart rate variability, HFnu and delta power bands across the first three sleep cycles.Eight patients suffering from severe SAHS before and after 4 ± 3 years of nasal CPAP treatment were matched with fourteen healthy controls. Sleep EEG and ECG were analysed to obtain spectral sleep and HRV components. Coherence analysis was applied between HFnu and delta EEG variability were decreased in untreated patients while gain values normalized in treated patients. In patients before and during long-term CPAP treatment, phase shift and delay between modifications in HFnu and delta EEG variability did not differ from controls but were not different from zero. In healthy men, changes in cardiac vagal activity appeared 9 ± 7 minutes before modifications in delta sleep.Sleep characteristics and spectral HRV components were similar between untreated patients, treated patients and controls, with the exception of decreased Rapid Eye Movement duration in untreated patients. Coherence and gain values between HFLong-term nasal CPAP restored, in severe SAHS, the information between cardiovascular and sleep brainstem structures by increasing gain, but did not improve its tightness or time shift. Increased cardiovascular events reportedly occur in the early morning hours ,2. InterThe causal link between sleep apnea-hypopnea syndrome (SAHS) and cardiovascular morbidity has remained controversial for many years ,7 but thIn patients suffering from SAHS, continuous positive airway pressure (CPAP) treatment improved sleep architecture ,18, cardNo studies have investigated the impact of long-term CPAP treatment on spectral HRV components across sleep stages, or the evolution of the link between HRV and deep sleep across the night in severely apneic patients treated by CPAP, in comparison to healthy controls.2) but when expressed in normalized units (nu) they may reflect more accurately the relative influence of one system versus the other on HRV  and treated patients [ 10-4 Hz] compared to healthy controls vs . Moreover, untreated patients showed decreased coherence and gain values in comparison to controls . During long-term CPAP treatment, gain values between HFnu and delta signals were similar to controls while coherence values were decreased . Phase shift between occurrence of modifications in HFnu and those in the delta power band did not differ between untreated apneic patients and controls  or between CPAP treated patients and controls . Similar results were obtained for the delay between occurrence of modifications in both signals and are shown in Figure panel c. Additionally, standard deviations of the phase shift and delay were larger than the corresponding mean values for untreated and treated patients. In healthy controls, modifications occurrence in HFnu preceded the occurrence of those in the delta power band with a phase advance corresponding to 9 ± 7 minutes.The frequency of the maximum of the cross-spectrum between HF1,33 ± 0,5 10-4 Hz1,33 ± 0,5 10-4 Hz1,33 ± 0,5 10-4 HzOur study revealed that long-term use of nasal CPAP ameliorates some parameters reflecting the relationship between heart and brain during sleep in patients suffering from SAHS. Furthermore, in SAHS, the link between cardiac vagal influence and delta sleep is altered even in the absence of significant differences in mean spectral HRV components between apneic patients and healthy men.nu and those in delta power bands, suggests that CPAP can not completely reverse the impairment of the relationship between heart and brain during sleep seen in patients suffering from SAHS. However, improved parameters of the relationship between cardiac sympatho-vagal balance and delta sleep, such as the gain between both signals, probably may contribute to the prevention of cardiovascular events in severely apneic patients treated with long-term CPAP.Therefore, the permanence of altered parameters such as the tightness of the link related to coherence values, or such as the phase shift between modification occurrences in HFOur patients suffering from severe SAHS showed decreased REM duration compared to healthy controls. Other sleep parameters did not differ from controls. Sleep characteristics of CPAP treated patients were similar to controls.Our results were in accordance with the sleep architecture of patients suffering from moderate-to-severe SAHS, treated by an oral placebo versus by nasal CPAP for 6 to 12 months . The autA previous study comparing sleep quality between sham-CPAP and effective-CPAP treatment in patients suffering from severe obstructive sleep apnea syndrome (OSAS) showed an absence of difference in sleep parameters between sham-CPAP and effective-CPAP treatment after seven days of treatment . NeverthOlder publications showed increased REM duration as well as deep sleep after the first night of nasal CPAP treatment ,52. ThesIn contrast with some data in the literature ,12,54, onu (hence lowered LF/HF). Indeed, assessment within sleep stages revealed that treated SAHS patients exhibited consistent trends towards greater HFnu (lower LF/HF ratio) in each sleep state. Thus, we hypothesize that long-term nCPAP use could increase the cardiac vagal predominance of HRV.Our SAHS patients treated by nCPAP did not differ significantly from healthy controls in terms of mean HRV components. Nevertheless, in treated SAHS compared to healthy subjects, there were strong trends towards lower absolute HRV values and increased HFnu values were decreased while HFnu was increased compared to patients with moderate-to-severe SAHS before treatment [nu, HFnu, and the LF/HF ratio during spontaneous and deep breathing were similar to baseline values implying the absence of increased relative cardiac vagal predominance induced by CPAP treatment [Only a few studies evaluated the impact of long-term CPAP treatment on cardiac sympatho-vagal activity ,20,25,26reatment . In contSimilar controversy was reported during the first days of CPAP treatment use in SAHS patients ,28,55. Onu, which reflects cardiac vagal predominance, tended to be greater compared to healthy controls without reaching statistical significance, a trend that was apparent whether we assessed HRV within individual sleep states or assessed the first 3 cycles of the night as a whole.Thus, numerous studies reveal that vagal predominance of the cardiac sympatho-vagal balance was induced by CPAP treatment in comparison to severely apneic patients before treatment ,20,55 buDuring the first three NREM-REM cycles, the LF/HF ratio did not differ from one in CPAP treated patients. This result indicates that none of the sympathetic and vagal branches of cardiac sympatho-vagal activity predominated, in contrast to the relative sympatho-vagal influence in healthy controls. Moreover, the absence of relative sympathetic or vagal predominance is found during REM sleep and wakefulness in patients treated by CPAP. This could imply that CPAP treatment increased cardiac vagal activity in contrast to the relative sympathetic activity. Indeed, CPAP treatment improved baroreflex sensitivity in SAHS ,57. Durinu and occurrence of those in the delta power band did not differ from controls, but showed larger fluctuations around the mean and did not certainly differ from zero. In agreement with our previous paper [nu and those in delta power band. This may be due to a more irregular breathing. In patients treated with long-term nCPAP, we observed decreased coherence and similar gain values in comparison to healthy controls. Moreover, the variability of the phase shift or the delay between HFnu and delta modifications was decreased in comparison with untreated apneic patients, but phase shift and delay were not different from zero.In accordance with previous reports , our patus paper , SAHS alThus, long-term nCPAP increases the strength of the interaction between the relative cardiac vagal predominance of HRV and delta sleep EEG but does not enhance the tightness of this link. CPAP treatment could ameliorate the quality of the information between brainstem cardiovascular and sleep structures through a regularisation of breathing and, as a result, an increase in the gain values and also a decrease in the variability of the time delay between modifications in cardiac sympatho-vagal influence and delta power band during sleep. Other non-linear biomathematical models that are specifically developed to determine the exact phase shift and the information flow of ECG-EEG signals, as phase slope index or Granger causality, could be applied to confirm our results .nu and delta indicated a lengthened mean sleep cycle duration and that the time at which both signals shared a maximum of power was later than that of healthy controls. Then, mean delay between modifications occurrence of HFnu and delta signals was later in untreated and treated patients in comparison to healthy men.Decreased frequency of the maximum of the cross-spectrum between HFOur patients were controlled with healthy men for age, nicotine, alcohol and caffeine consumptions. Moreover, they did not suffer from other pathologies than severe SAHS. They had no present or past medical condition that would modify cardiovascular or cerebral activities. They took no illicit drugs, psychotropic or somatic medications, or over the counter agents that could interfere with cardiac activity or sleep. Presumably, this “pure” sample does not reflect the general middle-aged population. The results are also limited by the small size of our groups. On the other hand, we can also ensure that comorbidities did not influence our analysis of relationship between cardiac vagal influence and delta sleep in SAHS and its adequate CPAP treatment. Nevertheless, we were not able to match BMI for patients and controls. The influence of BMI on HRV is controversial ,62. UsinOur data confirm alterations of the relationship between cardiac sympatho-vagal activity and delta power in patients suffering from severe SAHS, even in the absence of cardiovascular events or significant changes in HRV components. Moreover, new findings of this study reveal that long-term nCPAP treatment could ameliorate the interaction between heart and brain during sleep, and thus, avoid cardiovascular pathologies, even if CPAP treatment does not cure SAHS.The authors declared that they have no competing interest.FJ is the principal investigator of the study and participated to each step of this research. CK performed statistics. JL provided his help for the data collection and the interpretation of the results. AN, PV and PL contributed to the study design and the interpretation of the results. All authors read and approved the final manuscript.Research reported in this paper was supported by the Belgian National Fund for Scientific Research and the Erasme Academic Hospital – ULB.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2466/13/29/prepub