MicroRNAs (miRNAs) are a class of endogenous small regulatory RNAs. Identifications of the dys-regulated or perturbed miRNAs and their key target genes are important for understanding the regulatory networks associated with the studied cellular processes. Several computational methods have been developed to infer the perturbed miRNA regulatory networks by integrating genome-wide gene expression data and sequence-based miRNA-target predictions. However, most of them only use the expression information of the miRNA direct targets, rarely considering the secondary effects of miRNA perturbation on the global gene regulatory networks.We proposed a network propagation based method to infer the perturbed miRNAs and their key target genes by integrating gene expressions and global gene regulatory network information. The method used random walk with restart in gene regulatory networks to model the network effects of the miRNA perturbation. Then, it evaluated the significance of the correlation between the network effects of the miRNA perturbation and the gene differential expression levels with a forward searching strategy. Results show that our method outperformed several compared methods in rediscovering the experimentally perturbed miRNAs in cancer cell lines. Then, we applied it on a gene expression dataset of colorectal cancer clinical patient samples and inferred the perturbed miRNA regulatory networks of colorectal cancer, including several known oncogenic or tumor-suppressive miRNAs, such as miR-17, miR-26 and miR-145.Our network propagation based method takes advantage of the network effect of the miRNA perturbation on its target genes. It is a useful approach to infer the perturbed miRNAs and their key target genes associated with the studied biological processes using gene expression data.The online version of this article (doi:10.1186/1471-2105-15-255) contains supplementary material, which is available to authorized users. MicroRNAs (miRNAs), a class of ~22 nt endogenous small regulatory RNAs, can induce the degradation or translational repression of mRNA transcripts through sequence-specific binding to their 3’-UTRs , 2. To dTo study miRNA functions, biologists usually overexpress or knockdown specific miRNAs in cells and observe their impacts on cellular states and functions , 6. The In this study, we proposed a network propagation based method (NP-method) to identify the perturbed miRNA regulatory networks from the gene expression data. It used random walk with restart , 19 in gp-value for each miRNA. The software is freely available at ;Let each target be the RWR seed at each time and calculate the corresponding ;Sort this score vector in descending order and sort targets accordingly, then get a target rank NPESs, then get a new score vector ;Start from the first target in the rank and add the rest one by one to compose new RWR seed sets and calculate the corresponding NPES and the LE targets of the miRNA  to generate null distribution of the NPES for each miRNA.To avoid producing bias towards the miRNAs with large target set, we performed a permutation-based statistical analysis to normalize the p-value for the score of each miRNA, which is the proportion of obtaining NPES in the null distribution not less than the one actually observed [p-values of all miRNAs with the Benjamini & Hochberg method [NPES as a z-score:Subsequently, we computed the empirical observed . We implg method  using a Here the mean and standard deviation were calculated from the null distribution. Then the scores of different miRNAs were comparable, larger score implied the miRNA took more responsibility for the observed gene expression changes and should be more important for the studied biological process. We finally ranked miRNAs according to the normalized scores.We compared NP-method with two other methods on predicting the perturbed miRNAs. One is the popular gene set enrichment analysis (GSEA), which determines whether an a priori defined set of genes shows statistically significant, concordant differences between two biological states or phenotypes . We usedNPESzscore in NP-method, the normalized enrichment score in GSEA and GR.GSEA generated by the GSEA software), and compared these methods using the putative rank of the experimentally perturbed miRNAs. If the desired miRNA is ranked at the top, it implies the corresponding method can predict well enough. While analyzing the gene expression data from CRC patient, we used the area under the receiver operating characteristic (ROC) curve, named AUC, to evaluate the prediction of cancer associated miRNAs. Larger AUC means better prediction [During the analysis of gene expression data coming from miRNA overexpression or knockdown experiments, we sorted miRNAs in descending order according to the normalized scores  targets, which are most likely regulated by the perturbed miRNAs in the specified condition and give rise to the observed gene expression changes. Take the CRC dataset GSE18625 for example, it found 49 LE targets for miR-145 in the transfected DLD-1 cells. Among them the n cancer , FSCN1 aarcinoma , 35. Henarcinoma , which iNPESs of the miR-34a targets. According to the multi-target-based NPESs (red dots), we extracted 36 leading-edge targets that appear at and before the peak point. In the figure we see a special LE target (CUX1), whose fold change is small (marked in a red circle) but NPES is relatively large. To illustrate the network perturbation effect of this target gene, we investigated its surrounding gene regulatory network  but three downstream genes  had remarkable fold changes. Besides, CUX1 is a homeodomain transcriptional regulator known to be involved in the development and cell cycle progression, and its activity is associated with increased migration and invasiveness in numerous tumor cell lines including HCT116 or resistance to apoptosis in pancreatic cancer [KIF23, ECT2 and RACGAP1 play important roles in the cell cycle and cell proliferation [It is known that miRNAs tend to fine-tune the expressions of genes , 14, andk Figure C, where c cancer , 40. Andferation , 42. TheSince NP-method can identify key target genes of miRNAs, exploring the similarities and differences among the key targets of the same miRNA under different situations can further help to understand roles of miRNAs in different context. Besides, it is said that the influence of miRNA perturbation on gene expression is time-dependent . To checNPES represents to what degree the miRNA-induced network perturbation can explain the gene differential expression levels, so we checked the NPES of miR-124 at every time point and found it got the maximum at 24 h . Besides, there were seven common genes shared by every time-point’s LE target set. These genes should be regulated by miR-124 all the time after its transfection. They are CDK4, CD164, AMMECR1, RPIA, FAM177A1, RRBP1 and MBOAT5. The fold change patterns of these genes look very similar , it is a type I receptor for the TGFB family of signaling molecules and has been found inducing apoptosis through activating SMADs and MAPKS in tumor cells [From the results  to infer the perturbed miRNA regulatory networks using the differential expression information of global gene network. It executes random walk with restart (RWR) from the miRNA targets in the gene regulatory network to model the intracellular propagation effect of the miRNA perturbation, and meanwhile adopts a forward searching strategy to find the leading-edge targets that are principally regulated by the perturbed miRNAs and result in the observed global gene expression changes.CUX1 in HCT116 cells. Besides, the analysis of time-course gene expressions from the miR-124 transfected cells revealed that the influence of miRNA perturbation in cells might be time-dependent and our method was more suitable for analyzing the perturbation effect at early time than other methods. In brief, NP-method can help to uncover the perturbed key miRNA regulatory networks in cellular processes of interest.The analyses of the miRNA perturbed cell line data demonstrated that NP-method could detect perturbed miRNAs from gene differential expression profiles better than GSEA and GR.GSEA. Except for the prediction of pivotal miRNAs, another advantage is to extract the context-specific leading-edge targets for miRNAs at the same time. Even those low-key but functional targets, whose differential expressions are not much prominent but their down-stream gene expressions are significantly changed in response to the miRNA perturbation, can be discovered by our method. For example the miR-34a regulates NPES). The top-ranked miRNAs are more likely to cause the observed gene differential expressions and are considered more important for the studied cellular process. In the future, we will take the miRNA cooperative regulation into account and try to infer the combination of miRNAs for better deciphering the miRNA-mediated cancer pathologies.When analyzing the gene expression data of CRC patients, NP-method predicted the disease associated miRNAs better than other methods, which again proved its efficiency. And based on the results we successfully built a key miRNA regulatory sub-network that should be perturbed and play important functions in colorectal cancer. However, it is known that cancers are usually caused by multiple factors not just a single molecular deregulation like a miRNA overexpression or inhibition, so exploring the synergetic effect of a miRNA group should be more reasonable and meaningful. In this work, the NP-method considered the miRNAs or miRNA families as independent determiners of global gene expression changes and prioritized them according to the estimated network perturbation effect score . Figure S1. Size of LE target subsets of the experimented miRNAs. Figure S2. Score and leading-edge targets of miR-124 at seven time points. Blue dot represents the absolute value of gene expression fold change, which is normalized by the maximum of all genes; Red dot stands for the NPES generated by NP-method, and the peak value is the optimal score and those targets appearing at and before this point are the leading-edge targets. (PDF 216 KB)Additional file 1: Table S1: Putative ranks of experimentally perturbed miRNAs using different r in NP-method. Results of analyzing the six miRNA perturbed cell line datasets.(XLSX 128 KB)Additional file 2:Results of analyzing the time-course gene expression dataset and the original gene expression fold changes.(XLSX 1 MB)Additional file 3:Results of analyzing the colorectal cancer dataset, miR2Disease supported cancer miRNAs and the original gene expression fold changes.(XLSX 406 KB)Additional file 4:


Motor cortex excitability was found to be changed after repetitive transcranial magnetic stimulation (rTMS) of the temporal cortex highlighting the occurrence of cross-modal plasticity in non-invasive brain stimulation. Here, we investigated the effects of temporal low-frequency rTMS on motor cortex plasticity in a large sample of tinnitus patients. In 116 patients with chronic tinnitus different parameters of cortical excitability were assessed before and after ten rTMS treatment sessions. Patients received one of three different protocols all including 1 Hz rTMS over the left temporal cortex. Treatment response was defined as improvement by at least five points in the tinnitus questionnaire (TQ). Variables of interest were resting motor threshold (RMT), short-interval intra-cortical inhibition (SICI), intracortical facilitation (ICF), and cortical silent period (CSP).After rTMS treatment RMT was decreased by about 1% of stimulator output near-significantly in the whole group of patients. SICI was associated with significant changes with respect to treatment response. The group of treatment responders showed a decrease of SICI over the course of treatment, the group of non-responders the reverse pattern.Minor RMT changes during rTMS treatment do not necessarily suggest the need for systematic re-examination of the RMT for safety and efficacy issues. Treatment response to rTMS was shown to be related to changes in SICI that might reflect modulation of GABAergic mechanisms directly or indirectly related to rTMS treatment effects. Tinnitus is associated with neural changes in both the auditory pathway and in non-auditory brain areas . TinnituRepetitive transcranial magnetic stimulation (rTMS) as a therapeutic intervention consists of non-invasive repeated stimulation of neocortical areas for hundreds of times per daily sessions via principles of electromagnetic induction. The TMS coil is placed on the subjects’ head over the target area and short-lasting high-intense currents in the coil produce a strong magnetic field (up to 1.5 T), which passes largely undistorted through the scull and induces neuronal depolarisations in the underlying cortical area. rTMS leads to long-term depression- or potentiation-like neuroplastic changes . Low-freIn addition TMS can also be used as a diagnostic tool for the assessment of motor cortex excitability by quantifying contractions of peripheral muscles induced by stimulation of the corresponding motor cortex representation. Assessment of motor cortex excitability in a longitudinal study design before and after a specific therapeutic intervention (e.g. multiple sessions of rTMS) enables to describe treatment-related neuroplastic changes. It has been shown, that rTMS over the temporal cortex can induce changes in motor cortex excitability. In a sham-controlled study 27 healthy subjects showed decreased motor evoked potential amplitudes and delayed cortical silent period (CSP) after five days of 1Hz rTMS of the right temporal cortex . FurtherIn conclusion, these studies highlight the existence of cross-modal plasticity in rTMS studies in tinnitus or with temporal stimulation. Evidence for functional connectivity between temporal and motor cortex comes from neurophysiologic and brain metabolism studies ,18. In tAll participants gave written informed consent after a comprehensive explanation of the procedures. All studies whose data contributed to this analysis were approved by the Ethics Committee at the University of Regensburg. All experiments were conducted in accordance with the last revision of the Declaration of Helsinki.Measurements of cortical excitability were performed in 116 patients (84 (72.4%) males; 49.2 ± 12.5 21–83) years) with chronic tinnitus (duration 90 ± 94 (2–476) months). 28 (25.2%) out of 111 patients (data of 5 patients not available) reported a purely left-sided, 21 (18.9%) a purely right-sided tinnitus, and 62 (55.9%) patients described their tinnitus as bilateral or originating within the head. Tinnitus distress was assessed by the German version  of the T–83 yearsThe therapeutic intervention consisted of 10 rTMS sessions on consecutive weekdays. Treatment effects were evaluated by changes in TQ between the first (day 1) and the last day of treatment (day 12). Motor cortex excitability was examined on the first day before treatment and on the last day after rTMS. We analyzed longitudinal data of 116 patients participating in three different treatments studies ,22. PatiFor measurement of cortical excitability, participants were seated in a reclining chair. TMS was delivered by two Magstim 200 stimulators  connected via a Bistim module to a figure-of-eight coil (double-circular-70-mm coil). The coil was held tangential to the skull and with the handle pointing backwards and about 45° away from the midline. The optimal coil position for stimulation was defined as the position above the left motor cortex for eliciting MEP of maximal amplitude in abductor digiti minimi muscle with a slightly supra-threshold stimulus. Once this position was found, it was marked on a scientific head cap and the coil was held in this position by the investigator.Motor evoked potentials (MEPs) of the abductor digiti minimi of the right hand were recorded with surface electrodes. The analogue signal was registered, band-pass filtered between 20 Hz and 10 kHz, then digitised at a frequency of 5 kHz and analysed off-line. Resting motor threshold (RMT) was determined as the lowest stimulation intensity that evoked in at least four out of eight consecutive trials a MEP of at least 50 μV in the resting abductor digiti minimi . Active Short-interval intra-cortical inhibition (SICI) and intra-cortical facilitation (ICF) were measured with a paired-pulse TMS protocol . The intCortical silent period was measured in 10 trials  in the moderately active abductor digiti minimi muscle on the non-rectified recording of every individual sweep and then averaged . ParticiStatistics are based on retrospective analysis. We were interested if changes of motor cortex excitability were related to rTMS treatment per se and to rTMS induced clinical response. Thus, we did an analysis of variance with treatment as within-subjects factor (first vs. last day of treatment) and treatment response as between-subjects factor (responder vs. non-responder). Treatment response was defined as change in TQ of at least 5 points ,28. If aAs we had four variables of interest , significance threshold was set to a Bonferroni corrected level (0.05/4 = 0.0125). For post-hoc tests significance threshold was set to 5%. Statistical analyses were performed with SPSS 18.0.0 . As the focus of this analysis were rTMS induced effects on cortical excitability we abstain from reporting treatment efficacy data which have already been published elsewhere ,22,29,30Firstly, rTMS resulted in a near significant decrease in RMT (reduction by 1% stimulator output) in the whole study population (Table We found no significant effects of treatment (pre vs. post rTMS). For RMT there was a tendency towards a reduction  but this effect did not reach the Bonferroni-corrected significance level. There was no main effect of group (responder vs. non-responder) either. With respect to the group x treatment interaction there was a significant interaction effect between the change in SICI and the clinical response to rTMS  for the whole group. Since stimulation intensity in most rTMS treatment studies is adjusted to the resting motor threshold (RMT) several earlier studies investigated whether rTMS treatment changes the RMT -16,31-42Treatment responders as identified by a 5-point reduction in the TQ showed increased SICI at the beginning of the treatment and a reduction over the course of the treatment. Non-responders showed the reverse pattern. Because of the lack of a placebo group we cannot definitively conclude that the observed reduction of SICI during treatment in the responder-group is related to successful rTMS treatment. Theoretically the SICI change could also reflect tinnitus reduction alone, independently of the kind of intervention. However the fact that the responder and non-responder group also differed in their baseline values of SICI makes the latter explanation extremely unlikely.A relationship between treatment response and a reduction of SICI has not yet been reported before. The discrepancy to previous results may be related to statistical power since the here presented data are from the biggest sample so far. Two studies in healthy controls found no effects of temporal rTMS on SICI ,15; in aNevertheless, our findings of altered SICI add further evidence to the notion of cross-modal plasticity of motor cortex in context of stimulation of non-motor areas. Considered mechanisms as indicated by studies in synaesthesia and sensory deprivation (e.g. deafness or blindness) may take place via multi-sensory association areas, via direct cortico-cortical connections, or via subcortical interplay of the corresponding sensory systems at the thalamic level -47. In tBeside anatomical considerations of cross-modal plasticity, our findings can also be discussed in the context of trans-synaptic chemical signalling. Both antidepressant effects  and tinnOn a functional level auditory-motor connectivity might be related to functions of speech or music perception. The motor theory of speech perception links vocal tract gestures to the perception of speech . Beat inThe counterpoint to the big sample size are the missing control groups and the retrospective analysis. Therefore further prospective studies including control groups are needed before firm conclusions about the specifity of the observed effects for rTMS treatment of tinnitus can be drawn. Moreover consecutive, daily measurements would enable to better characterise the time course of changes in excitability.Nevertheless the presented data add to the literature that rTMS of non-motor areas can induce changes in motor cortex excitability. RMT changes during rTMS treatment seem to be negligible and do not affirm the potential need for re-evaluation of the stimulation intensity during treatment for efficacy and safety issues. SICI changes might reflect modulation of GABAergic mechanisms directly or indirectly related to rTMS treatment effects.No author has to declare competing interest except the receipt of third-party funds from the Tinnitus Research Initiative for the conduction of clinical trials of rTMS in tinnitus.MS, ML, TK, EF, PGS, PE, GH, BL have made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data. All authors have been involved in drafting the manuscript or revising it critically for important intellectual content. MS, TK, RR, and BL have given final approval of the version to be published. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors read and approved the final manuscript.


Skin antisepsis is an important measure to prevent postoperative infections. It is known that wound infections infringe the well-being of the patient and prolongs the rehabilitation significantly. At the same time additional costs will be caused in the health care system. Alcohol-based solutions containing the active ingredients chlorhexidine gluconate [CHX] or octenidine dihydrochloride [OCT] have a residual antimicrobial effect on skin. This may re-sult in a better preventative outcome than alcohol alone.The aim of the presented study was to compare the immediate and long-term efficacy of CHX and OCT.We performed a study on the skin on the arm of 20 healthy volunteers in a cross-over design based on DGHM-standard method 13 and measured the colony forming units (cfu) of the resident skin flora after the application of the products according to Williamson and Kligman (1965). The cfu were determined directly after the application and after 24 h on 5 consecutive study days. The calculated log reduction factors were statistically evaluated with the student’s t-test.Both solutions showed as expected a significant any quick reduction of the resisdent skin flora and a long-term effect over 24 h. For OCT a statistically significant superior long-term effect after four applications was determined .The presented data show that alcoholic solutions with octenidin dihydrochloride and chlorhexidine gluconate show a comparable efficacy on the resident skin flora. Alcohol-based preparations with the additional active octenidin dihydrochloride are a valid alternative for skin antisepics to the world-wide broadly used CHX-based products.F. Brill Grant/Research support from: partially by Schülke & Mayr, Germany., N. Radischat Employee of: Schülke & Mayr, Germany., P. Goroncy-Bermes Employee of: Schülke & Mayr, Germany., J. Siebert Employee of: Schülke & Mayr, Germany.


As a single receptor, the neonatal Fc receptor (FcRn) is critically involved in regulating albumin and IgG serum concentrations by protecting these two ligands from degradation. In addition to these essential homeostatic functions, FcRn possesses important functions in regulating immune responses that are equally as critical and are increasingly coming to attention. During the first stages of life, FcRn mediates the passive transfer of IgG across the maternal placenta or neonatal intestinal walls of mammals, thereby conferring passive immunity to the offspring before and after birth. In fact, FcRn is one of the very few molecules that are known to move from luminal to serosal membranes of polarized cells that form epithelial barriers of the lung and intestines. Together with FcRn’s recently explored critical role in eliciting MHC II presentation and MHC class I cross-presentation of IgG-complexed antigen, this renders FcRn capable of exerting broad and potent functions in regulating immune responses and immunosurveillance at mucosal sites. Further, it is now clear that FcRn dependent mucosal absorption of therapeutic molecules is a clinically feasible and potent novel route of non-invasive drug delivery, and the interaction between FcRn and IgG has also been utilized for the acquisition of humoral immunity at mucosal sites. In this review, we begin by briefly summarizing the basic knowledge on FcRn expression and IgG binding, then describe more recent discoveries pertaining to the mechanisms by which FcRn orchestrates IgG related mucosal immune responses and immunosurveillance at host–environment interfaces within the adult organism. Finally, we outline how the knowledge of actions of FcRn at mucosal boundaries can be capitalized for the development and engineering of powerful mucosal vaccination strategies and novel routes for the non-invasive delivery of Fc-based therapeutics. Fcgrt gene, is an MHC class I-like transmembrane protein that requires non-covalent association with β2-microglobulin (β2-m) for proper functioning. In its best known function, FcRn binds to the CH2-CH3 interface of the IgG Fc region in a 2:1 stoichiometry  signals into the host intramural immune system. To mirror the situation in humans, these studies utilized humanized mice in which a human FcRn transgene (together with human β2-m) was expressed in a mouse that was devoid of mouse FcRn  demonstrated that after intravenous injection of anti-OVA IgG and subsequent oral feeding of fluorescently labeled OVA, OVA-IgG immune complexes (ICs) formed within the gastrointestinal lumen and were transported in an FcRn dependent manner into the LP where the antigen–antibody complex was taken up by CD11c+ DCs which, upon MHC II presentation of OVA epitopes, were then able to induce an antigen-specific CD4+ T-cell response  large intestine and induce IL-12 production in an FcRn-dependent manner. Further, survival analysis indicated that CRC patients with ≥10 FcRn+CD11c+ cells per observed area had significantly longer survival times over a 70-month follow up than did those with <10 FcRn+CD11c+ cells in the tumor microenvironment  and the Fc fragment of mouse IgG1, showing that bioactive Fc-Epo fusion protein was successfully absorbed FcRn dependently via the respiratory epithelium . In subsin utero, these studies clearly imply that pathogenic antibodies can also cross the maternal–fetal barrier. That this is indeed the case has been shown in a murine model of fetal and neonatal immune thrombocytopenia (FNIT) in which FcRn-mediated transplacental transfer of maternal pathogenic antibodies results in the destruction of platelets in the neonate  deficiency, in which glycosaminoglycan storage begins in prenatal life, it was shown that a GUS-Fc-fusion protein, infused into the mothers, was transported across the placenta into the fetus, leading to reduced lysomal storage disease only in those mice whose mothers were administered GUS-Fc . While t neonate  but satu neonate . Apart f neonate . Collectin vivo for both bacterial and viral agents, where FcRn-mediated translocation of systemically administered pathogen-specific IgG to the mucosal site of pathogen entry within the gastrointestinal or genital tract was identified as the key mechanism responsible for protection  glycoprotein gD and the Fc domain of IgG2a together with the adjuvant CpG, protected wild type, but not FcRn knockout, mice after intravaginal challenge with virulent HSV-2 and this immunization strategy induced an efficient mucosal and systemic antibody response as well B- and T-cell immunity (In addition to passive immunity, it has already been demonstrated immunity . Similarimmunity . Of partimmunity , 56. Finimmunity . Table 1+ and CD8+ T-cell responses and thus exert broad functions in immune surveillance and immune homeostasis. Critically, these pathways have already been shown to have important pathophysiological relevance for infectious diseases and cancer development along mucosal barriers. Exploitation of the receptor–ligand interaction between FcRn and IgG has already been successfully used for the development of novel mucosal vaccination strategies as well as the non-invasive delivery of Fc-fusion proteins. Thus, the roles that FcRn plays in guiding the transport of IgG across epithelial barriers and regulating the recycling of monomeric IgG away from a degradative fate in lysosomes and preserving IgG half-life or alternatively directing IgG containing ICs to the antigen presentation machinery present enormous therapeutic opportunities for translation (Table In the recent past, the view of FcRn has changed from a receptor, which contributes to passive immunity via transcytosis of IgG molecules to that of an active signaling and trafficking receptor that is deeply involved in antigen presentation pathways. It is now clear that FcRn inherits a fundamental role in classical MHC II presentation of exogenous antigen as well in the cross-presentation of exogenous antigen on MHC I molecules. The pleiotropic functions enable FcRn to elicit antigen-specific CD4on Table . Our incThe 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.


Image-guided prostate interventions often require the registration of preoperative magnetic resonance (MR) images to real-time transrectal ultrasound (TRUS) images to provide high-quality guidance. One of the main challenges for registering MR images to TRUS images is how to estimate the TRUS-probe-induced prostate deformation that occurs during TRUS imaging. The combined statistical and biomechanical modeling approach shows promise for the adequate estimation of prostate deformation. However, the right setting of the biomechanical parameters is very crucial for realistic deformation modeling. We propose a patient-specific deformation model equipped with personalized biomechanical parameters obtained from shear wave elastography to reliably predict the prostate deformation during image-guided interventions. Using data acquired from a prostate phantom and twelve patients with suspected prostate cancer, we compared the prostate deformation model with and without patient-specific biomechanical parameters in terms of deformation estimation accuracy. The results show that the patient-specific deformation model possesses favorable model ability, and outperforms the model without patient-specific biomechanical parameters. The employment of the patient-specific biomechanical parameters obtained from elastography for deformation modeling shows promise for providing more precise deformation estimation in applications that use computer-assisted image-guided intervention systems. Prostate cancer is the most common noncutaneous cancer and the second leading cause of cancer death in menet al.In the last decade, prostate deformation modeling has been investigated as a solution for image-guided prostate interventions. Two main methodological categories have been studied: biomechanical modeling and statistical modeling. Because it is easy to implement, the mass-spring model (MSM) initially came to dominate the biomechanical models3et al.et al.On the other hand, statistical modeling methods have been proposed for predicting the deformation by analyzing a set of training data and generating a statistical model, which can be applied in real-time applications. Dam in vivo biomechanical parameters of the prostate and assigned them to an FE model to form a patient-specific deformation model.Nevertheless, combining a statistical model with a biomechanical model with patient-specific parameters shows promise for achieving an adequate model of prostate deformation. Because collecting a sufficient number of representatives of the population as training data is essential for building an effective statistical model but collecting a large number of patient data is often difficult in clinical practice, biomechanical modeling shows promise as a way to generate sufficient data for statistical analysis. On the other hand, to achieve a physically appropriate biomechanical model, determining the biomechanical parameters of prostate tissue is importantThe main contribution of this research is to investigate the impact of employing patient-specific biomechanical parameters obtained from SWE data for prostate deformation modeling, by comparing the performance of deformation model constructed with and without patient-specific biomechanical parameters. We further implemented a non-linear elastic material model to describe the non-linear stress-strain behavior of the prostate when undergoing TRUS-probe-induced deformations. Details are described in the method section. The experimental results show that the patient-specific deformation model outperforms the model without patient-specific biomechanical parameters in terms of deformation estimation accuracy.The 2D prostate phantom images with and without probe induced deformation are shown in To illustrate the effect of biomechanical parameter setting on the deformation estimation, we systematically perturbed the biomechanical parameters of inner and outer prostate with positive and negative offsets from the SWE measures for the model-guided MR-TRUS registration on all the patient data. For the perturbing of outer prostate, the parameter of inner prostate was fixed as SWE the measurement, whereas the outer prostate parameter was similarly unchanged while perturbing the parameter of inner prostate.p-values of two-tailed student tests) between the registration results of using changed tissue parameter and unchanged SWE measurement.It can be observed from We proposed a deformation model for estimating patient-specific prostate deformation during image-guided interventions. We employed shear wave elastography to obtain the patient-specific biomechanical parameters. Coupled with a non-linear elastic material model, these biomechanical parameters are beneficial for generating a realistic deformation model for practical applications. The experimental results demonstrated the superior performance of our proposed deformation model over the traditional model without patient-specific biomechanical parameters in terms of deformation estimation accuracy.Precise measurements of tissue properties have been shown to be crucial for a realistic biomechanical modeling7in vivo personalized biomechanical parameters can be acquired by readily accessible SWE, our patient-specific deformation modeling method should be easily adapted for these clinical applications to improve their accuracy of deformation estimation.Although the deformation model we developed in this study was primarily designed for the purpose of estimating prostate deformation, it is a general modeling method that makes use of SWE data and has the potential for being applied in other clinical applications for the following reasons. First, shear wave elastography is widely utilized in clinical diagnosis and has become a valuable method for imaging the prostate, liver, thyroid, breast, etc., complementing conventional ultrasound and MR3 in x-, y- and z-direction. The 3D TRUS images were obtained by a Mindray DC-8 ultrasound system  with an integrated 3D TRUS probe. The TRUS voxel size was 0.5 × 0.5 × 0.5 mm3 in x-, y- and z-direction. The shear wave elastography images were acquired using a Supersonic Aixplorer  ultrasound system. The Supersonic Aixplorer provides the Q-Box tool which can calculate the average Young’s modulus within an operator selected region. The average Young’s moduli of inner and outer prostate were obtained separately by sampling several Q-Boxes within the corresponding glands. To ensure the accuracy, the Q-Boxes were selected by experienced doctors.Experiments were carried out on the datasets obtained from a prostate phantom and twelve patients with suspected prostate cancer at the First Affiliated Hospital of Sun Yat-Sen University. The study protocol was reviewed and approved by the Ethics Committee of Sun Yat-Sen University and informed consent was obtained from all patients. The methods were carried out in accordance with the approved guidelines. A set of MR, TRUS, and SWE data were acquired from each patient. The MR images were applied to construct the geometric model of the prostate; the SWE data were employed to obtain the patient-specific biomechanical parameters; the TRUS images that showed the deformed prostate were used to evaluate the deformation estimation performance of the proposed deformation model through the MR-TRUS registration. The T2-weighted MR images were acquired using a 3.0 Tesla Siemens TrioTim MR-scanner  with a 32 channels body coil. The MR voxel size was 0.625 × 0.625 × 3.6 mmOur modeling framework consisted of two steps see . We firsWs is formulated as:When undergoing image-guided interventions, the prostate often deforms, primarily due to the insertion of the TRUS probe. Previous mass-spring and finite element methods modeled using linear elastic models have not been able to simulate such large deformations. In our implementation, we assumed that the involved tissues are elastic/hyperelastic isochoric materials and employed a neo-Hookean model to formulate the biomechanical behaviors of the prostate. Many studies have applied this model to predict the non-linear stress-strain behaviors of materials undergoing large deformationsJel is the elastic volume ratio, G is the shear modulus and K is the bulk modulus. The Jel can be calculated from the elastic deformation tensor Fel:where Cel = FelTFel, is the elastic right Cauchy-Green deformation tensor and u is the displacement vector and I is the unit matrix). On the other hand, with the strain energy density Ws, the second Piola-Kirchhoff stress can be calculated as:where u can be figured out from the S via Ws. Combining G and K) and the boundary conditions . Details of the implementation follow.Finally, the displacement G and bulk modulus K to the solver is essential. These two moduli are material-dependent and can be calculated by Young’s modulus E and Poisson’s ratio ν:Assigning an accurate shear modulus andν, we assume that the prostate is nearly incompressible and simply set it to be a constant 0.495E for a specific patient can be challenging, especially because different regions of the prostate have different biomechanical characteristics. In addition, these properties vary from patient to patientG and bulk modulus K were calculated and assigned to the neo-Hookean model.For the Poisson’s ratio To obtain the information for the geometric model, we segmented the prostate and bladder gland using data from MR images via an interactive segmentation software: SmartPaintThe boundary conditions define the external forces and restrictions exerted on the prostate during TRUS-guided interventions. The insertion of the TRUS probe is the main cause of prostate deformation. To realistically model the interactions between the TRUS probe and the prostate, the front end of the TRUS probe was reconstructed based on its physical shape. Then the prostate and 3D TRUS probe models were set as mechanical contact pairs in our implementation. Other boundary conditions, such as the patient’s position and the pelvic bone surface, have little influence on the deformation of the prostateWe employed COMSOL, an FE analysis software, to calculate the deformation of the prostate. We input the governing equations and the material-dependent parameters to the non-linear FE solver embedded in COMSOL. In our implementation environment, each deformation modeling took about 15 seconds to calculate the mesh displacement.in vivo. To this end, we further employed the statistical modeling method to analyze the deformations under different probe insertion situations. To collect data that was representative of the population for statistical modeling, we conducted Q  biomechanical modelings for each patient using the same anatomic model and biomechanical parameters, but different settings for the probe insertion conditions. Through such personalized biomechanical modeling, each modeled result was able to efficiently represent the patient-specific prostate deformation induced by a particular insertion of the TRUS probe. Afterwards, the patient-specific deformation model for each patient was calculated by statistically analyzing the Q modeled deformation instances using PCA method.Although the anatomic geometry and biomechanical parameters are patient-specific for deformation modeling, the conditions of the TRUS probe insertion are very difficult to measure Two experiments were conducted to validate the impact of patient-specific biomechanical parameter setting on deformation modeling.To analyze the effect of the biomechanical parameter setting on the accuracy of deformation modeling, a validation experiment was first carried on one prostate phantom Model 053-AEF . Two specific TRUS image sets were acquired for the quantitative analysis on the efficacy of using patient-specific biomechanical parameters. First, one TRUS prostate image set, denoted as P1, without probe-induced deformation was obtained and manually segmented as the basis for the parameterized deformation modeling. The other TRUS image set (P2) was acquired with the in-plane deformation caused by the probe insertion. The position of the TRUS probe was recorded by the electromagnetic tracking sensor attached to the probe, thus the probe movement information can be accurately calculated. Our objective is to illustrate that whether the deformation modeling result on P1 by adopting patient-specific biomechanical parameters is similar to the realistic prostate deformation on P2. To quantitatively demonstrate the influence of the biomechanical parameter setting for deformation modeling, eight uniformly sampled elasticity values from a specific range  of tissue parameter as recommended in Hu’s work, 230 kPaWe employed the patient-specific deformation model to perform model-guided MR-TRUS registrationThe target registration error (TRE)How to cite this article: Wang, Y. et al. Patient-specific Deformation Modelling via Elastography: Application to Image-guided Prostate Interventions. Sci. Rep.6, 27386; doi: 10.1038/srep27386 (2016).


In utero hematopoietic stem cell transplantation (IUHCT) is an attractive approach and a potentially curative surgery for several congenital hematopoietic diseases. In practice, this application has succeeded only in the context of Severe Combined Immunodeficiency Disorders. Here, we review potential immunological hurdles for the long-term establishment of chimerism and discuss relevant models and findings from both postnatal hematopoietic stem cell transplantation and IUHCT. Central tolerance: An immune mechanism for specificity of the immune response set up in the thymus. Functional central tolerance prevents T cells with a high affinity to “self” from exiting into the periphery.Dominant tolerance: A tolerance mechanism that can override other tolerance mechanisms such as Tregs (regulatory T cells) or myeloid-derived suppressor cells with capacity to suppress effector mechanisms of other cells.Regulatory T cells: Specialized CD4+FoxP3+ T cells that inhibit proliferation and effector functions of other immune cells via several mechanisms. Most studied for their effect on CD8+ T cells but potentially also important for regulation of other effector cell types.Clonal deletion in reference to exhaustion: A type of terminal differentiation in T cells (CD4 and CD8) elicited by continuous presence of antigen. T cells gradually lose functionality in a stepwise, strictly controlled process and sometimes die as a function of continued antigen presence. Partial exhaustion is sometimes reversible.Anergy: Unresponsiveness of T cells previously stimulated with their cognate antigen in the absence of an appropriate second signal. Anergic T cells do not execute normal effector function.Peripheral tolerance: Immune protocols that mediate specificity of the immune response, other than central tolerance. All the following can be considered peripheral tolerance:In utero hematopoietic stem cell (HSC) transplantation is not a standard clinical approach, but with greater understanding of the immune system and its development, as well as the disease processes that are optimally treated in utero, the procedure may become more widely used. Stem cell transplantation, as any other transplantation is subject to potential rejection reactions by the hosts' immune system that recognizes the tissue antigens of a different genetic makeup as “foreign” and elicits an immune attack. This attack, if not properly controlled by immune-suppressants, can lead to graft damage and ultimately graft loss. Transplantation of hematopoetic stem cells in utero seeks to take advantage of the early developmental stages of the fetal immune system and elicit a dampened immune rejection or perhaps achieve full graft tolerance. While attractive in theory, the practical outcomes of in utero hematopoetic stem cell transplantation have been disappointing for a multitude of reasons. Here we review the complex issues pertaining the immune system that have bearing on in utero hematopoetic stem cell transplantation for a general clinical audience.The main function of the immune system is to fight off infection of a huge variety of pathogens. To successfully fight “foreign” invaders without damaging the host, the immune system has to sort through enormous antigenic diversity to recognize the difference between “self” vs. “non-self.” Allogeneic transplants (solid-organ and cell transplants) are not “self” from the recipient perspective and hence are attacked by the recipient immune system  necessitating the administration of immunosuppressants. However, successful HSC transplants will actively participate in the immune system as they give rise to all white cells. Additionally, the special situation of HSC transplantation in the context of the fetal immune system during IUHCT warrants specific considerations of the developing recipient environment to ensure graft survival.Several fundamentally different processes establish necessary self-tolerance and are also involved in  graft-tolerance: central tolerance, exhaustion/peripheral deletion and regulatory T cells (Treg).Ex vivo stimulation of a central tolerant T cell population with self-antigens does not result in activation or proliferation. Central tolerance is effective but not complete and a few self-reactive T cells can migrate into the periphery despite a functioning central tolerance mechanism . While central tolerance and T regs both mediate non-reactivity to self, the mechanisms by which tolerance is established are very different. There is another distinction between the two mechanisms: Central tolerance is dependent on localization to the thymus while Tregs are mobile, and when transferred to another host, can and will perform appropriate suppressive function if properly stimulated and maintained  all the mechanisms discussed above result in non-reactivity of the responder T cells  of the thymus supports the early stages of T cell development, when thymocytes are selected for ability to interact with self-MHC molecules. Cells that survive this positive selection process travel on to the inner thymus (medulla), where negative selection against self-tissue-specific antigen recognition takes place (self -peptide-tolerance).Lymphoid progenitor cells migrate during embryonic development in several discrete waves, coordinated with organogenesis , endothelial cells, and dendritic cells. Thymic epithelial cells are the main cell type interacting with and providing selective clues to T cells Figures , 3. BaseInitial stages of T cell selection ensure that the resulting pool can successfully “communicate” with cells of the host. To ensure proper communication, T cells must be equipped to engage in productive interactions with proteins that constitute the communication system, the MHC proteins. The cortical thymus selects for this function in a process termed positive selection: T cells with low-affinity interaction of T cell receptors with peptide-MHC complexes receive survival signals while T cells with no or too low affinity engagement die by neglect  and the utilization of unconventional molecular pathways by APC. These pathways ensure unique self-peptide repertoires for both the negative and positive selection steps, with peptides derived from the thymus itself, from the periphery and different developmental stages, including some from stages other than the current developmental state. This remarkably diverse repertoire is ensured by (1) promiscuous gene expression by mTEC, (2) specific antigen processing features including proteasome composition and protease sets, (3) intracellular antigen sampling via autophagy of thymic epithelial cells and (4) extracellular antigen sampling provided by immigrating dendritic cells (DC) that sample the periphery. mTEC express hundreds of self-antigens that are otherwise expressed only in strictly regulated spatial or temporal contexts. In mTEC, proteins are translated for the sole purpose of undergoing degradation, after which they are loaded onto MHC molecules. Regulation of this complex and unique process is still poorly understood but the recent discovery of the transcriptional regulator Aire has shed some light on this process , a subset of CD4+ T cells defined by intranuclear expression of the transcription factor FoxP3. Thymic Tregs (tTregs) develop during thymic T cell selection from CD4+ clones with moderate-to high-affinity for self-antigens. Peripheral Tregs (pTregs) arise from mature, naïve CD4+ T cells in the periphery when they encounter antigen under certain conditions   tissue cells to which they have been exposed sufficiently early in fetal life” -peptides by recipient APC. This type of presentation will be continuously functional throughout the lifetime of the graft, as recipient APC circulate and pick up antigen. First observed in solid-organ transplantation, the kinetics and contribution of direct vs. indirect antigen presentation pathways are much less understood in HSC transplantation and IUHCT.In animals or humans with mixed bone-marrow chimerism, both donor and recipient HSC can theoretically give rise to the different blood products of all blood lineages. To generate peripheral T cells, donor T cells must be positively and negatively selected in the thymus Figures , 3. The in vivo and in MLR accompanied by an exhaustion mechanism that limits reactivity of the recipient-reactive donor cells that escape thymic selection.In a SCID patient with a haploidentical fetal liver and thymus transplant, T cell clones were established after 11 years of healthy life of this patient (no indications of GVHD)  genes. In those mice, stimulation of allo-responses occurs only via professional APC while in wild-type mice, both professional APC and non-professional cells such as endothelial cells can present antigen. At day 60, cytotoxicity, recall responses, and memory formation were impaired in wild type mice experimentally. In contrast, in mice devoid of minor HC genes in the non-hematopoietic compartment, none of these features of exhaustion appeared and host-reactive T cells remained fully functional even after 60 days, suggesting that antigen presentation by non-professional APC is responsible for allo-specific T cell exhaustion. These observations strongly indicate that recipient-reactive donor T cells can escape thymic clonal deletion but will acquire an exhausted phenotype in the periphery due to continued presence of “their” antigen in the context of non-professional antigen-presenting cells. The extent to which this is the underlying explanation for T cell exhaustion in the context of IUHCT remains to be determined.Information about the mechanism of T cell exhaustion can be gleaned from the study of HSC transplantation in the setting of chimerism with non-hematopoietic recipient cells serving as potential APC is similar to IUHCT. Indeed, the antigenic source responsible for donor T cell exhaustion was addressed in a mouse model of allogeneic bone marrow transplantation. Here, a clinical scenario of non-myeloablative transplantation and delayed lymphocyte infusion was modeled  and successful participation of graft-derived cells in the selection and maturation processes to form fully differentiated cells. Failure of any of these requirements may limit engraftment or lead to poor long-term graft function but the participation of each of these components in the final phenotype is sometimes difficult to assess. In IUHCT, the presence of the maternal immune system adds another layer of complexity, the effects of which are only partially understood.Current observations in IUHCT models suggest that successful engraftment starts in the fetal liver from which the cells migrate into the bone marrow. In mice, CXCR4 chemokines follow an SDF-1a chemoattractant gradient for fetal-liver to fetal bone marrow migration. Migrating HSC also express matrix metalloproteinases (MMP-2 and MMP-9) at their leading cellular edge, facilitating degradation of extracellular matrix during migration toward marrow, and they secrete collagen and metalloprotease inhibitors to modify the niche. Hematopoietic stem cells used for IUHCT should express the necessary migratory molecule repertoire and other factors of fetal liver HSC to allow engraftment.Facilitation of engraftment was further addressed in an IUHCT model in which donor bone marrow was stimulated with vascular endothelial growth factor and stem cell factor  are detectable in fetal livers as early as 7.5 weeks of gestation, in thymus at 12.5 weeks, and spleen and blood at 14.5 weeks  antibodies are transported to the placenta via an FcR-mediated process (Malek et al., In IUHCT, maternal anti-donor antibodies have been shown to play an important role in long-term chimerism maintenance: Merianos et al. demonstrated that maternal sensitization during IUHCT and subsequent transfer of alloantibodies via maternal breast milk induces an adaptive immune response in mouse pups which leads to graft rejection (Merianos et al., Many maternal cell types contribute to microchimerism of the fetus; cells of myeloid and lymphoid lineages have been identified as well as hematopoietic progenitors (Jonsson et al., Maternal cells with anti-donor specificity can also directly inhibit the establishment of chimerism in IUHCT (Nijagal et al., Altered maternal fetal cellular trafficking in humans has been reported after fetal surgeries, preeclampsia and pregnancy termination but potential roles for these insults in the limitation of chimerism has not been determined (Jeanty et al., in vitro and in vivo experiments are needed to dissect the mechanisms of tolerance and rejection for this potentially important clinical procedure.The fetal immune system is a complex system that changes dramatically as a function of developmental stage, and to patterns of exposure to alloantigens. Given that direct query of the immune response after IUHCT is difficult, well-designed The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The ectomycorrhizal (ECM) symbiosis connects mutualistic plants and fungal species into bipartite networks. While links between one focal ECM plant and its fungal symbionts have been widely documented, systemic views of ECM networks are lacking, in particular, concerning the ability of fungal species to mediate indirect ecological interactions between ECM plant species (projected-ECM networks). We assembled a large dataset of plant–fungi associations at the species level and at the scale of Corsica using molecular data and unambiguously host-assigned records to: (i) examine the correlation between the number of fungal symbionts of a plant species and the average specialization of these fungal species, (ii) explore the structure of the plant–plant projected network and (iii) compare plant association patterns in regard to their position along the ecological succession. Our analysis reveals no trade-off between specialization of plants and specialization of their partners and a saturation of the plant projected network. Moreover, there is a significantly lower-than-expected sharing of partners between early- and late-successional plant species, with fewer fungal partners for early-successional ones and similar average specialization of symbionts of early- and late-successional plants. Our work paves the way for ecological readings of Mediterranean landscapes that include the astonishing diversity of below-ground interactions. Evaluating the extent and functions of the ecological links that soil biota create among terrestrial plants is a fascinating challenge in ecology. In temperate and boreal forests, ectomycorrhizal (ECM) symbiosis ecologically binds together 3% of terrestrial plant species and more than 6000 filamentous fungal species. Below-ground mycelia directly connect short roots to soil resources, and provide pathways for reciprocal nutrient fluxes and water exchanges among associated individuals . ECM symSarcodes sanguinea and Neottia nidus-avis  or small  numbers of partners.Ectomycorrhizal fungal species also display large variation in the number of associated plant species, e.g.CM trees  whereas us Alnus . Phylogeth Alnus . HereaftBipartite network: a network linking two distinct sets of nodes (with no links among nodes of the same set). Figures 1 and 2A illustrate the ectomycorrhizal (ECM) ecological network linking plant species with fungal species. Bipartite networks are also termed 2-mode networks or bimodal networks.Common mycorrhizal network (CMN): below-ground network where fungal mycelia physically connect roots of different plant individuals.k):Degree  corresponds to the number of its symbiotic partners. It measures its interaction specialization.Ecological mycorrhizal network: mutualistic interaction network linking together plant and fungal entities  able to establish a mycorrhizal connection in at least one ecological context and during one ontological stage. The ecological ECM network studied here at the species level  is qualitative (binary links) and only informs on the potentiality of two species to interact.Interaction specialization: tendency to interact with few or lot of partners. A species that interacts with many species (high degree k) is termed an interaction generalist and a species that interacts with few species (low degree k) is termed an interaction specialist. In the case of the ECM ecological network, fungal interaction specialization is often called host-specificity of the fungal species in mycological literature.Modular network: a modular network is made of subsets of nodes highly connected between them and poorly connected to others. These subsets are called modules or clusters. Modularity in an ecological network may reflect ecological  and evolutionary  processes.c):Partner specialization , where the mean is taken over the set of its partners. Here, the partner specialization of a focal plant species is the mean number of host plant species of its fungal symbionts . The average of nc over plant species of same bipartite degree recovers the standard bipartite degree correlation (sometimes termed connectivity correlation).l):Projected degree . Here, the projected degree of a focal plant species is the number of other plant species sharing fungal partners with it.Projected network: 1-mode network built from a bipartite network, by considering only nodes of one set, and linking two nodes if they share at least one neighbor in the bipartite network ; the links of a projected network are also termed indirect links, mediated by nodes of the other set. A bipartite network is associated with two projected networks. Here, the plant projected network links ECM plants through their shared fungal partners :Projected weight . Here, the projected weight of a focal plant species is the number of fungal species shared with other plants, where each fungal species is counted as many times as it indirectly links the focal species to another plant species. The projected weight of a focal plant species is in general different from its projected degree nl, but related to its partner specialization nc and its bipartite degree nk according to n= 1+(sn/kn).cin vitro cultivation, fungal species may appear less host-specific than they really are due to cryptic diversity, and determinants of fungal host-specialization are still controversial  constructed from a unique qualitative dataset that exhaustively assembled field and molecular records on ECM plant–fungi associations in the island of Corsica, France. ECM links between plant and fungal species are considered at the scale of the whole island. More specifically, our aim was to understand the potential of ECM symbiosis for creating fungi-mediated ecological interactions  between plant species, and reciprocally between ECM fungal species by means of shared host plants. These indirect interactions among species of the same kind (plants vs. fungi) mediated by species of the other kind constitute Our study investigated three main questions. First, whether there is a trade-off between the number and the specialization of fungal species associated with a plant species; second, whether plant species associated with fewer fungal species in the bipartite network are linked to fewer plant species in the projected network; and finally, when the ecological strategies of plants are considered, whether early- and late-successional species display different interaction patterns, in either the bipartite network or the projected plant network.2 of mountainous territory. It is the sole island in the Mediterranean basin presenting large surfaces of well-preserved native ECM forests . We also excluded mycoheterotrophic and mixotrophic plants  or because their ECM fungal communities are insufficiently documented  from 61 informative analyzed datasets extracted from either fruitbody surveys or DNA belowground studies . The corresponding 16 × 411 binary matrix of association has value 1 at position  if fungal species f has been reported to be an ECM symbiont of plant species p, and 0 otherwise . The number of plant species that are linked with a given fungal species f is called its bipartite degree fk. Low k values characterize interaction-specialist species  will be used to refer to the realized biotic part of plant niche, and not to any theoretical niche of ECM plants in the Corsican region. The fungal species linked with the plant species p may also be linked with other plant species, and the number of plant species that are linked through one or more of these shared fungal partners is called the projected degree of the plant species, pl. These links constitute the projected network of plant species (or “plant projected network”). We likewise defined the projected degree of a fungal species, fl, as the number of associated fungal species in the projected network, i.e., linked through plant species to the focal fungal species f .We built a bipartite network including the 16 plant species and 411 ECM fungal species  links of the plant species p to other plant species is called its weight ps in the plant projected network . Likewise, the total number of plant-mediated links of a fungal species f to other fungal species is its weight fs in the fungal projected network.A plant species can interact with another plant species pl represents the number of plant species to which p is linked in the plant projected network, ps represents the total number of links established via fungal species with these plant species. The contribution to ps of a plant species is simply the number of ECM fungal species that it shares with p. Therefore, the ratio of ps to pl measures in an integrated way the redundancy of fungal species in establishing links between p and other plant species.While pk) and the specialization of the fungi  with which it associates. Indeed, a plant species associated with many specialized fungal species (high pk and partners with low fk) may be as well-linked (same ps) with as many other plant species (same pl) as a plant species associated with few generalist fungal species (low pk and partners with high fk). To explore this question, we introduced an additional quantity, pc, measuring the average specialization of the fungal partners of a given plant species p. It is defined as the mean degree (in the bipartite network) of the fungal partners of the plant species, p, where the average runs over the set of these fungal partners. We then devised two null models to analyze the relationships between pc and pk (see below). A summary of the different network-related notions and their notation is given in the Glossary (Box Box 1).If the number of links of a given plant species to other plant species is selected for, then we may expect a trade-off for the plant between its specialization  considering independently each pair of plant and fungal species and drawing a link with a probability equal to the density of links  observed in the real network. As a consequence, the mean values of plant and fungal bipartite degrees are the same in this null model 1 and the real network.pk and pc values obtained with this null model were compared with the distribution and relationships obtained in another null model, in which the bipartite degree of each plant or fungal species is exactly the same as in the real network, but the links between species were randomized (null model 2). Both null models were generated using the permat functions within the R package Vegan with  preserving column and row sums . Finally, observed values of pc and pk were compared with their statistics obtained in the null model 2 in order to detect whether the actual network differs from a random network in which levels of species specialization  were identical to those observed in the real data.The distributions and relationships between row sums . Using t network . We pres∗ 411 fungi), our analysis focused on a plant perspective as more information  is available for each plant, allowing a more powerful statistical analysis. The fungal-perspective analysis is presented in Supplementary Figure Given that the association matrix is highly asymmetric (16 plants pk), their weights in the projected network (ps) and average numbers of hosts of their fungal symbiotic species (pc) in the sets of early-stage and late-stage plant species, using Mann–Whitney non-parametric tests. We measured modularity using the simulated annealing algorithm implemented in Netcarto  fungal communities associated with Betulaceae  and Cistaceae  to rich ECM fungal assemblages  associated with Quercus suber and Quercus ilex evergreen oaks . There was no significant correlation between the bipartite degree kp of a plant species and the interaction specialism cp (average number of host species) of its associated fungal species .Among the 16 plant species, association patterns varied from species-poor (low pk) and fungal (fk) bipartite degrees were much higher in the real network than in the simple random model . We then constrained the null model so that the degree of each plant (pk) and fungal species (pk) is the same as in the real network . The simulation revealed a strong negative correlation between plant degree (pk) and mean interaction specialism of fungal partners (pc) . Hence, pk and fk values impose a constraint on the structure of the network which a priori results in a strong negative correlation between number of associated fungal species and degree of specialization of these fungal species . In the real network, the absence of correlation between the bipartite degree pk of a plant species and the average number of hosts pc of its associated fungal species is unexpected given the distribution of pk and fk values.The variances of the plant  shows that the value of pc deviates from the null model for more than half of the species . Four plant species  host significantly more specialist fungal species than would be expected by chance  while the four Fagaceae species  and Corylus avellana host significantly more generalist fungal species than expected by chance .Comparing the real network and the null model with constrained pl = 15; Table 1). Indeed, the number of plant species to which a plant species is linked in the projected network varies from 11  to all other 15 species . Behind the tendency of all plant species to saturate their projected network, there is wide variation in the number of ECM-mediated links (two-step links) that a given plant species makes with all other plant species , so that the neighborhood of Halimium halimifolium in the projected network includes all the plant species of the present survey. In contrast, Betula pendula symbiotic links do not saturate the plant–plant projected network even though its 46 associated ECM fungal species establish 214 fungi-mediated links.Ten plant species are linked to all other plant species . In our analysis, these two endemic alders establish no ECM fungi-mediated links with Betula, Corylus, Populus, or Salix.Endemic alders, ps values, taken as the weight of the plant species in the projected network), ranging from 36 ECM-mediated links for A. alnobetula to 474 links for Q. ilex  that a given plant species makes with all other plant species (pl/ps (ratio of the projected degree pl to the number of comprised links ps) ranges from 11/36 = 0.306 for A. alnobetula, to 15/474 = 0.032 for Q. ilex (Table 1). These results indicate a lower redundancy of the ECM-mediated links of A. alnobetula to other plant species as compared to Q. ilex.The redundancy Figure 3 and Table 1, with a maximal degree pk = 46 for the set of early-stage species and a minimal degree pk = 67 for the set of late-stage species). On average, early-stage plant species associated with five times fewer fungal species . They also present five times fewer fungi-mediated links to other plant species in the projected network  and five times higher pl/ps ratios . Nevertheless, their associated fungal species did not differ in their partner specialization pc  from those associated with late-stage plant species .Early-stage vs. late-stage plant species displayed contrasted distributions of their numbers of associated fungal species . Fewer ECM fungal species (62) than expected by chance (108.52 ± 4.95 under null model 2) interact with both early- and late-stage plant species (p < 10-3). More ECM fungal species (37) are exclusively associated with early-stage plant species than expected by chance  given the number of partner species. On the contrary, the number of ECM fungal species (272) exclusively associated with late-stage plant species is close to the value expected by chance . These results show that during ecological successions, late-successional vegetation stages accumulate rich ECM communities comprised of specialist fungi that differ in composition compared to the communities associated with early-successional stages  between early- and late-stage plant species, (ii) among early-stage plant species, and (iii) among late-stage plant species, against the sharing calculated in the null model with constrained M = 0.458, null model = 0.362 ± 0.002). Three modules included only early-stage plant species  and the three others included the late-stage species .We further used the modularity of the whole network to characterize the sharing of ECM symbionts among early- and late-stage plant species. We detected six modules in a significantly modular system (A. glutinosa (early-stage) and Q. ilex (late-stage) illustrates the contrast between early- and late-stage plant species association patterns . Fungi-mediated interactions of Q. ilex are (i) quantitatively highly variable  and (ii) qualitatively more numerous toward Fagaceae . All but two fungal species associating with Q. suber also associate with Q. ilex in Corsica (Inocybe fibrosoides is strictly associated with Q. suber and Boletus pulverulentus is strictly associated with Q. suber and C. avellana). Similarly, A. glutinosa presents a high variation in the number of fungi-mediated links toward other plant species, ranging from one  to 12 (A. cordata). Both A. glutinosa and Q. ilex show higher numbers of fungi-mediated links toward plant species belonging to their own genus  and, for Q. ilex, to its family .Comparing In this paper we analyzed the main properties of the ECM bipartite network linking host plants and fungal species in Corsica and deciphered ECM network structure in an ecological perspective. We did not detect significant correlation between the specialization of plant species and the specialization of their symbionts across the whole plant dataset, but rather observed decoupled patterns of symbiont diversity  and the specialization of these symbionts (average number of plant partners of these symbionts). Our results show that early- and late-successional plant species share fewer fungal symbionts than expected by chance, with early-successional species presenting fewer links caused by fewer fungal partners than late-successional plant species, and no difference in average specialization of fungal symbionts of early- and late-successional plant species.Figure 3A). In other words, the analysis of the whole dataset shows no trade-off between plant specialization and the specialization of its symbionts. As a consequence, the number of fungal partners of a plant species (pk) poorly reflects the number of plant species with which it has fungi-mediated indirect interactions (pl).In our analysis, plant species hosting the richest ECM fungal assemblages associate with fungi whose host ranges are similar to those of plants with few fungal partners . Interestingly, all but two fungal symbionts of Q. suber are also associates of Q. ilex. Only two of the 133 fungal symbionts of Q. suber are host-specific, while 39 species out of the 197 fungal associates of Q. ilex are host-specific (Table 1). This pattern may result from a mix of phylogenetic and ecological proximity. Indeed, the distribution and the ecological range  of Q. suber in Corsica tend to be included within those of Q. ilex  due to abundant plant–plant connections at genus , family . This tendency to saturate the projected network . As a consequence, similar numbers of neighbors in the plant projected network hide highly variable numbers of symbionts . The contrasted patterns observed for A. glutinosa and for Q. ilex illustrate this point. In our analysis, A. glutinosa saturates the plant–plant network based on only 54 fungal-mediated links whereas Q. ilex use 474 such links to do so . This result points out the composite nature of Alnus-associated communities, which are comprised of (i) fungal symbionts belonging to Alnus-associated fungal lineages , (ii) interaction specialists scattered across distant fungal lineages and (iii) a few generalist fungal species  and scattered montane ECM species , but no Betula, Corylus, Populus and Salix, remain in the landscape  a low dependence on water compared to other alders, Salix and Populus, and (ii) an ability to establish under Pinus, Abies, Fagus, and Quercus during secondary successions  has been hypothesized to increase physical networking and facilitate seedling establishment under pre-established trees in either conspecific  or mixed species , facilit forests  or the cenziesii .Beyond such fungi-mediated interactions between plant species, the ecological and evolutionary implications of the ability of an ECM plant species to interact with a high number of plant species remain undocumented. A saturated ECM projected network may increase fungal inoculum availability by maintaining compatible reservoirs on alternative hosts. This tendency to share symbionts with many other plants may enlarge the biotic component of the plant niche. Specifically, a high degree in the projected network may facilitate seedling establishment in vegetation of various stages and composition. Such plant-mediated facilitation mechanisms may have been particularly favored in Mediterranean ecosystems where summer drought drastically impacts tree recruitment . FurtherSalix and Alnus species are adapted to hydromorphic soils, where their host-specific fungal partners may exhibit adaptations to this specific abiotic environment.For fungal species, the ECM projected network provides a view of the pool of potentially interacting fungal species. As in plant-centered approaches, calculating the relative overlap in host plants between pairs of fungal species  would provide fungal species attributes. Mediterranean forests are generally dominated by locally monospecific tree stands . In thisFigure 4). Our data do not support the hypothesis that early-stage plants differ in the specialization of their symbionts from late successional species, but suggest that early-stage plants associate with significantly fewer ECM fungal species than late-stage plants. Additionally, long-lived late-successional plant species that dominate forest ecosystems for centuries allow a lasting fungal recruitment, which may entail the accumulation of pioneer ECM species. Interestingly, our dataset encompasses mostly species belonging to late-successional fungal genera (Russula, Boletus, Amanita, various “Aphyllophorales” including Bankeraceae and Hydnaceae; In our study, ECM association patterns significantly differed depending on the ecological strategy of their plant host. On average, early-stage plants had five times fewer fungal associates than late-stage ones (To the best of our knowledge, our work documents for the first time the below-ground fungal counterpart of the ecological strategies of ECM host plants. Despite the unprecedented rhythm of research describing ECM communities worldwide, their role in plant community dynamics in general, and in host coexistence processes in particular, remains largely unexplored, mainly because most ECM fungi are not cultivable. We assembled a large qualitative dataset to propose a systemic view of the interactions established by the tremendous ECM fungal diversity at the scale of a large Mediterranean island. The ECM symbiosis shapes ecological and evolutionary-based interaction modules, and can help us understand the below-ground niche differences between early- and late-stage plants. The huge diversity of symbionts contributes to saturate plant–plant networks through highly variable numbers of fungi-mediated interactions between plants. Assembling quantitative data of below-ground plant–fungi interaction is crucial to grasp the ecological dimension of these contrasted patterns.AT, FM, AL, FR originally formulated the idea, developed the methodology and performed statistical analyses. A-CM, J-MB, P-AM, FR generated data. AT, FM, P-AM, AL, FR wrote the initial manuscript. AT, FM, AL, A-CM, J-MB, M-AS, P-AM, and FR contributed to the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The purpose of the study was to assess the therapeutic effect of the angiogenesis inhibitor bevacizumab on plaques of various stages in rabbit models using contrast-enhanced ultrasonography (CEUS). Abdominal aortic atherosclerosis was induced in 55 rabbits. Thirty-six randomly selected rabbits were divided into 2 groups according to the timing of the bevacizumab injection: an early-stage plaque group (Group ESP) and a later-stage plaque group (Group LSP). The remainder were considered the control group. Standard ultrasonography and CEUS imaging of the abdominal aorta were performed. The animals were euthanized after CEUS, and plaque specimens were harvested for histological staining of CD31. The control group exhibited a substantially higher enhanced intensity, a higher ratio of enhanced intensity in the plaque to that in the lumen, and an increased number of CD31-positive microvessels in the plaque sections than Groups ESP and LSP . A higher enhanced intensity (P = 0.044), a higher ratio of enhanced intensity in the plaque to that in the lumen (P = 0.023) and more CD31-positive microvessels in the plaque sections (P = 0.006) were found in Group LSP than in Group ESP. Bevacizumab demonstrated more advanced inhibition of neovascularization in early-stage plaques in rabbits. Clinical studies have revealed that a vulnerable plaque is closely associated with the incidence of acute cardiocerebrovascular events136in vivo810121415Advances in contrast-enhanced ultrasonography (CEUS) have enabled the detection of neovascularization within atherosclerotic plaques In both Groups LSP and ESP, there were no differences in systolic and diastolic blood pressure, heart rate, total serum cholesterol (TC), triglyceride (TG), or low-density lipoprotein cholesterol (LDL-C) level before and after bevacizumab administration .All experimental rabbits developed multiple plaques in their abdominal aortas. The control group exhibited a higher intimal-medial thickness (IMT) than Groups ESP and LSP . StandarNo significant difference in plaque area, IMT or the maximum thickness of the plaques was observed between Groups ESP and LSP. The control group exhibited a higher IMT, maximum thickness of the plaques and a larger plaque area compared with Groups ESP and LSP .The plaques in the control group contained a greater number of CD31-positive microvessels compared with those in Groups ESP and LSP. A greater number of CD31-positive microvessels were detected in Group LSP compared with Group ESP  and 3. Nin vitro and animal studies have suggested that angiogenesis inhibitors can suppress plaque neovascularization and atherosclerotic progression617Many experimental VEGF is considered to be a potent regulator of neovascularization under both physiological and pathological conditions. Therefore, the inhibition of VEGF may eliminate plaque neovascularization and the consequent development of high-risk atherosclerotic plaques22in vivo because of its noninvasive, high spatial and temporal resolution and the properties of the contrast agent microbubbles, which behave as pure intravascular tracerset al.CEUS can be used to monitor real-time intraplaque neovascularization The present results demonstrate a more advanced inhibitory effect of bevacizumab on neovascularization in early-stage compared with later-stage plaques. This finding highlights the vital role of early anti-angiogenesis therapy for the treatment of vulnerable plaques. Indeed, the diffusion of oxygen and other nutrients is limited to 100 μm from the lumen of the blood vessel, which, in vasa vasorum-derived microvessels, is adequate to nourish the inner media and intimal layers. As the plaque enlarges, the ensuing hypoxia and/or inflammatory cell infiltrate promote intraplaque neovascularizationThe present data suggest that there are no changes in intraplaque neovascularization in response to different doses of bevacizumab. This unexpected finding differed from a previous report showing that VEGF inhibits intraplaque microvessels in a dose-dependent manneret al.Because the acoustic beam emitted from our probe could not completely penetrate the narrow intercostal spaces to provide valuable ultrasonoscopy, the cardiac function of rabbits was not assessed to identify any effects of bevacizumab on the heart. However, Pu in vivo.This study demonstrates that bevacizumab could inhibit neovascularization within atherosclerotic plaques. A more advanced bevacizumab inhibition of neovascularization in early-stage compared with later-stage plaques was observed, highlighting the vital role of early anti-angiogenesis therapy in the treatment of vulnerable plaques. CEUS imaging may be used to monitor these phenomena in real-time The experiment protocol was carried out in accordance with the Animal Management Rule of the Ministry of Health, People’s Republic of China  and was approved by the Scientific Affairs Committee on Animal Research and Ethics of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (China). Abdominal aortic atherosclerosis was induced in 55 Japanese white rabbits  provided by the Laboratory Animal Centre of Wuhan Research Institute of Biological Products through a combination of a cholesterol-rich diet and aortic balloon endothelial denudation.On the 4th week after balloon injury, all of the experimental rabbits underwent standard ultrasound imaging of the aorta to confirm the success of animal models of atherosclerosis by detecting local thickening of IMT or small plaques. Then, thirty-six rabbits selected randomly were divided into 2 groups according to the timing of the bevacizumab injection: an early-stage plaque group , and a later-stage plaque group . The remaining rabbits were considered the control group . Group ESP was randomized into two subgroups based on the dose of bevacizumab: ESPh , and ESPl . Similarly, Group LSP was randomized into LSPh  and LSPl . On the 5th, 6th, 7th and 8th weeks after balloon injury, the rabbits in Group ESP were treated four times with a transvenous injection of bevacizumab  during the ultrasound imaging. The arterial blood pressure and heart rate of each animal was monitored and recorded during both standard and CEUS imaging as described above. Standard ultrasonography and CEUS imaging of the abdominal aorta were performed as previously described.5 mL/kg After standard ultrasound imaging, the animal underwent CEUS imaging of the previously identified plaquesThe enhancement of the plaque after injection of the contrast material was quantitatively analysed offline as previously described−kt + B, where Y is the signal intensity at time t, k is a factor proportional to the transit time of the contrast agent, A is the derived peak signal intensity, and B is the intercept signal intensity at the origin of the curve (baseline intensity or background intensity)The abdominal aortic plaque and lumen signal intensity-versus-time curves during the process of enhancement were automatically produced and fitted to an exponential function: Y(t) = At·eRabbits were sacrificed via an intravenous injection of an excessive amount of 3% pentobarbital (3.0 mL/kg) after ultrasound imaging.After the abdominal aorta and right kidney artery were separated, previously identified plaques were harvested according to their distance from the right kidney artery measured using standard ultrasonography. All specimens were fixed in 2% paraformaldehyde for 24 h and embedded in paraffin. Subsequently, the tissue samples were serially sectioned (3 μm) and stained with haematoxylin-eosin for plaque morphometric evaluation and CD31  to specifically identify neovascularization. The plaque images were analysed with the Image Pro plus system . Plaque area, IMT and maximum plaque thickness were evaluated. Plaque area was calculated as the difference between the luminal area and the area delimited by the internal elastic lamina. The results were normalized to the total vessel cross-sectional area for each arterial section to eliminate variations due solely to vessel sizeRabbits were fasted over night before blood was drawn. Serum was separated by centrifugation and stored at −80 °C until analysis. TC, TG, and LDL-C levels were measured using an automatic biochemistry detection system before and after bevacizumab treatment.Data analysis was performed using SPSS software . All values are expressed as the mean ± standard deviation. Parameters assessed in the ESP, LSP and control groups were compared by analysis of variance. Significant differences between groups were assessed using the Scheffe F test for multiple comparisons. Significant differences were compared between each group before and after treatment with bevacizumab using a paired t-test. An independent-sample t-test was applied to identify significant differences between groups that received low-dose versus high-dose bevacizumab. A Pearson correlation was used to assess the linear association between enhanced intensity and the histological intraplaque neovascularization density. Differences with a p value of less than 0.05 were considered significant.How to cite this article: Li, Y. et al. The therapeutic effect of bevacizumab on plaque neovascularization in a rabbit model of atherosclerosis during contrast-enhanced ultrasonography. Sci. Rep. 6, 30417; doi: 10.1038/srep30417 (2016).


Cluster headache (CH) is a debilitating headache disorder with strong chronobiological features characterized by severe pain attacks often waking the patient from sleep. Hypocretin (HCRT) is involved in the regulation of arousal and pain and is produced in the hypothalamus. HCRT has been suggested to play a role in the pathology of CH.To investigate HCRT-1 levels in CH during active bout.CSF HCRT was measured in 12 chronic and 14 episodic CH patients during active bout, and in 27 healthy controls. Patients were characterized beyond the dichotomy of current diagnostic criteria (episodic/chronic) using a CH index reflecting total headache duration and their attack rhythmicity compared to HCRT concentrations.We found lower HCRT-1 levels in both chronic  and episodic cluster headache  compared with controls (430.96 pg/ml) and a tendency towards relatively higher values in chronic CH compared with episodic CH. A positive relationship between the CH index and HCRT concentrations was found in all patients . We also identified a tendency towards relatively higher HCRT concentrations in patients without chronological rhythmicity.This is the first report of lower HCRT-1 concentrations in CH suggesting that decreased levels may reflect an insufficient anti-nociceptive activity of the hypothalamus. The exact mechanism of the anti-nociceptive effect of HCRT is not known and requires further investigation. In conclusion, this study supports the hypothesis of a connection between arousal- and pain- regulation and the pathogenesis of CH.No conflict of interest.


Superposition and indistinguishablility between probability amplitudes have played an essential role in observing quantum interference effects of correlated photons. The Hong-Ou-Mandel interference and interferences of the path-entangled photon number state are of special interest in the field of quantum information technologies. However, a fully generalized two-photon quantum interferometric scheme accounting for the Hong-Ou-Mandel scheme and path-entangled photon number states has not yet been proposed. Here we report the experimental demonstrations of the generalized two-photon interferometry with both the interferometric properties of the Hong-Ou-Mandel effect and the fully unfolded version of the path-entangled photon number state using photon-pair sources, which are independently generated by spontaneous parametric down-conversion. Our experimental scheme explains two-photon interference fringes revealing single- and two-photon coherence properties in a single interferometer setup. Using the proposed interferometric measurement, it is possible to directly estimate the joint spectral intensity of a photon pair source. Nonlocal quantum interferences between strongly correlated photons are of fundamental interest not only for obtaining a deeper understanding of quantum mechanics, but also to explore the field of quantum information science. The observation of nonlocal two-photon interference effects is now considered as one of basic steps toward implementing photonic quantum information technologies. The fundamental principle underlying modern quantum optical technologies utilizing correlated photon pairs is that a photon-pair interferes with the pair itself, when the indistinguishability of two-photon probability amplitudes is guaranteed in coincidence detection. Representative two-photon quantum interference phenomena include the Hong-Ou-Mandel (HOM) interference effect arising from various conditions1245Since the original HOM experiment was performed, various types of HOM experiments have been performed to explore the relation between coherence and indistinguishability7891011In this paper, we present an experimental demonstration of the most generalized two-photon interference with photon pairs that are independently generated by spontaneous parametric down-conversion in the telecommunication wavelengths. The proposed two-photon interferometric scheme has the following properties. (a) The local behavior of individual single photons in a correlated pair at the BS is completely independent so that the measurement of photons has to be performed under strict local condition 1617V denotes the visibility of the coincidence fringe and To observe the nonlocal two-photon interference effect in an interferometer, it is required that the two interfering photons (two correlated photons) do not meet at the same BS simultaneously. Rather, the two photons have to be separated sufficiently far from each other to satisfy the Einstein locality condition, i.e., the travel time difference between the two photons must be longer than the coincidence time window320Now, we propose a conceptual schematic to explain the generalized nonlocal two-photon interference between separate photon pair sources, as shown in ϕ depends on the relative path-length difference between the short and long paths, it can be adjusted by varying the path-length difference, Since the phase factor The experimental setup to demonstrate the proposed two-photon interference is shown in Our experimental scheme is composed of two fibre interferometers as depicted in Correlated photons generated from a PPLN crystal do not overlap at the same BS. Individual photons from the two sources are combined at two spatially separated fibre BSs (FBS1 and FBS2) to interfere nonlocally. To obtain the full interferogram of the single-photon and the two-photon wave packet, ODL 2 is scanned in the range of single-photon coherence length for various positions of ODL1 within the two-photon coherence length, as shown in V represents the fringe visibility and For alignment of the interferometer, the HOM two-photon interference between independent photons from the two PPLN crystals was observed in the two-fold coincidence measurements with detectors D1 (D3) and D2 (D4) as a function of the path-length difference Upon introducing an unbalanced path-length difference using  and  , we can In particular, the analysis of the two-photon interference fringes obtained from the proposed interferometric measurement using the theoretical description of the joint spectral intensity of the photon pair source (see Methods for details) can yield the asymmetric bi-photon wave functions. The experimental results in In conclusion, we experimentally demonstrated nonlocal two-photon interference between pairs of photons in a fully unfolded and the most general form of the two-photon interferometer including the HOM scheme with non-overlapping two-photon amplitudes and involving spatially separated N00N-like two-photon states, which was carried out by employing two separate but identical photon pair sources. Nonlocal interference between photon pairs was observed even when two photons of a pair did not arrive at the same BS at the same time; this situation cannot be treated using the classical wave theory, and therefore, the situation has no classical analogy. The observed interference fringes simultaneously showed single-photon and two-photon coherence properties in a single interferometer setup. Using the proposed interferometric measurement, we can obtain the joint spectral intensity via the inverse Fourier cosine transform of the full interferogram. Quantum interferences between correlated photons can be utilized to explore quantum communication technologies such as entanglement-based quantum communication, single-photon quantum switching, and single-photon quantum routing by virtue of distributed nonlocal quantum correlations2324The full characterization of the photon pair source in our experiment can be obtained by simply measuring the single and coincidence counting rates as a function of the applied pump power. For an average pump power of 20 mW, the coincidence efficiency (coincidence to single ratio) was measured to be about 4.7% , and the coincidence to accidental coincidence ratio was about 2.68. The pair production probability per pulse is obtained by dividing the accidental coincidence by the actual coincidence ratio, which was estimated to be 0.37 per pulse in our experiment. For the pulse-pumped SPDC photons accidental coincidences are mainly caused by redundant multiple pair events per pulse.Since the two incoming photons have no time correlations, the HOM effect is rarely observed when the two photons arrive from each source simultaneously. In the experiments, when the two individual photons arrived at FBS1 (FBS2) at the same time, the coincidence counting rate between two detectors was reduced to 0.33 in raw visibility under perfect experimental conditionsi, and A, SB) or The quantum state of photon pairs from separate source  and   has the symmetrized bi-photon wave function, which is given as This result is similar to H. K. and H. S. M. conceived the project and designed the experiment. H. K. performed the experiment. S. M. L. contributed to the theoretical analysis. All authors discussed the results and contributed to writing the manuscript.