Discovery of communities in complex networks is a fundamental data analysis problem with applications in various domains. While most of the existing approaches have focused on discovering communities of nodes, recent studies have shown the advantages and uses of link community discovery in networks. Generative models provide a promising class of techniques for the identification of modular structures in networks, but most generative models mainly focus on the detection of node communities rather than link communities. In this work, we propose a generative model, which is based on the importance of each node when forming links in each community, to describe the structure of link communities. We proceed to fit the model parameters by taking it as an optimization problem, and solve it using nonnegative matrix factorization. Thereafter, in order to automatically determine the number of communities, we extend the above method by introducing a strategy of iterative bipartition. This extended method not only finds the number of communities all by itself, but also obtains high efficiency, and thus it is more suitable to deal with large and unexplored real networks. We test this approach on both synthetic benchmarks and real-world networks including an application on a large biological network, and compare it with two highly related methods. Results demonstrate the superior performance of our approach over competing methods for the detection of link communities. Many complex systems in the real world exist in the form of networks, such as social networks, biological networks, Web networks, etc., which are collectively referred to as complex networks. One of the main problems in the study of complex networks is the detection of community structure Although previous research towards community detection mainly focused on the community of nodes, several recent works begin to switch the attention to community of links Recently, a number of approaches to the detection of link communities in graphs have been proposed. For instance, Ahn et al. izθ’s, where izθ denotes the propensity of node i to have links in the z-th community. Then, they take izθjzθ as the expected number of links in the z-th community connecting nodes i and j. Finally, they fit the parameters of this model using a method of maximum likelihood evaluation based on an expectation-maximization algorithm.Moreover, in face of the good performance and sound theoretical principles, generative models form a promising class of techniques for identifying communities from networks. Techniques that are being actively researched and developed zω’s and izφ’s, where zω denotes the size of a link community z, and izφ describes the degree of importance when node i forms links in this community. Then, the expected number of links in the z-th community connecting nodes i and j is denoted by zφizφjzω. Compared with Ball’s model, here we introduce an additional set of parameters zω’s to characterize the sizes of different communities, aiming to make it more flexible when describing link communities. Thereafter, in order to fit the parameters of this model, we define it as an optimization problem based on squared loss, and solve it by using a technique of nonnegative matrix factorization (NMF). At last, we extend the above method by introducing a strategy of iterative bipartition, namely NMFIB, which can not only determine the number of communities all by itself but also get these results with a high efficiency. Therefore, this combined approach is better suited for discovery in unexplored and large networks. Also of note is that, this iterative bipartition process can be used to improve other model-based methods, such as Ball’s method.In this work, based on Ball’s model, we propose a new generative model to describe the community structure of links. This model is parameterized by two sets of parameters In this section, we first introduce a model for the description of link communities in networks, and then present a method based on nonnegative matrix factorization to fit the model parameters. Thereafter, we offer an example to illustrate the method. At last, we extend the above method to a new one that automatically determines the number of communities.n of vertices and m undirected edges. Assume that the links can be partitioned into c communities using a soft community membership variable R, where ijzR denotes the probability that link  belongs to community z, subject to zω’s and izφ’s, where zω denotes the size of community z and is defined as twice the expected number of links (or weight) in this community, and izφ denotes the probability that community z selects node i when it generates edges. Thus, we have We define the generative model of link communities, which produces networks with a given number i, j) can be generated as follows. First, we choose a community z randomly with an expected size zω. Then by using probabilities izφ, jzφ, community z selects nodes i, j as a pair. Consequently, the expected number of links in community z, that lies between nodes i and j, can be evaluated asBased on the above model, an edge  belongs to community z. Then we haveFormally speaking, assume that the community assignments are represented by a set of variables i, j) to community r satisfying r = argmaxz {ijzR, z = 1,2,…,c}, and then get the hard partition of links.As the soft membership of communities cannot be used directly, we can simply assign each link nn× can be achieved by optimizing (4). In the rest of this section, a method based on nonnegative matrix factorization (NMF) is developed to solve the optimization problem in (4).X, where izX is defined asWe first introduce an auxiliary matrix The loss function in (4) can be rewritten as a constrained nonnegative matrix factorization problem,F denotes the Frobenius norm, and X, using (5) zω can be given asλ is a hyper-parameter that reflects the importance of the hard constraints. Violation to stronger hard constraints incurs a higher penalty to the objective function. In our experiments, we first get an initial value of X0 by setting λ  = 0. Then we restart the optimization with X = X0 and let λ to be a relatively large number, e.g. 1000, to minimize the chance of violating the parameter constraints. The purpose of the initialization is to restrict the model search so as to start from some good approximation. Similar to other forms of NMF, the objective function in (9) is not convex w.r.t. X, so that it is computationally intractable to find a global minimum. Therefore, a heuristic, gradient descent strategy is adopted to search for local minima. This gradient descent strategy can be implemented in a multiplicative updating algorithm similar to the method for SNMF X is introduced to (9), resulting in the following equivalent objective function,It is nontrivial to directly optimize (6) with the hard constraints. We relax this optimization problem by introducing a penalty term that represents the hard constraints into the objective function, arriving at minimizing the following objective function,For any stationary state, we haveiz(X)iz  = 0, we have the following equation,Using complementary slackness condition (Θ)X:This leads to the following update rule for When the above iteration rule converges, the converged solution satisfies the Karush-Kuhn-Tucker (KKT) conditions The convergence of the iterative updating rules follows the theorem below.Theorem 1:The objective function O in (9) is non-increasing under the update rule in (10). O is invariant under these updates if and only if X becomes stationary.Proof: We adopt the auxiliary function approach used in Expectation-Maximization and NMF. The basic idea is to construct an auxiliary function X, then we are guaranteed to drive O(X) down.If we can minimize Note that,O(X). We can define the series of update rules as:X as in (10).So we get the update rule for AX in (10) is mc, where m is the number of edges and c is the number of communities. The time to calculate 1n(1nTX) is nc, where n is the number of nodes. The time of calculating X(TXX) is nc2, and the time of calculating 1n(1nTX)(TX1n(1nTX)) is also nc2. Consequently, the time to evaluate (10) once is O(mc+nc2), and hence the time complexity of our method is O(T(mc+nc2)), where T is the iteration number for convergence. Also, according to O(Tmc). Therefore, the time complexity of our method is competitive with that of Ball’s since nc is often competitive with m.Notice that, the time to calculate Here we depict the main idea of our method using a simple example, illustrated by G by optimizing (4), and get the best parameters of the model zω’s and izφ’s which are shown as zω’s and izφ’s, we can form the expected graphs of all the link communities in G according to (1), which are shown as G according to (2), which can be regarded as an ensemble of the expected graphs of all its communities, shown as G shown in First, we fit the expected graph to the given network c, i.e., the number of communities in a network. This is also a common drawback suffered by almost all methods based on generative models. The statistical model selection applied to generative models may, in principle, be able to find the number of communities in a consistent and satisfactory manner Nonetheless, the method mentioned above can still be improved. The main drawback is that, our model offers no criteria for determining the value of parameter c of communities is given, as large networks often have large values of c, the convergence rate of the core optimization algorithms  will necessarily become very slow. This is also an important limitation from existing model-based methods when dealing with large-scale networks in the real world.Furthermore, even if the number c  = 2, and then recursively subdivide the two parts. In dividing a subnetwork, we isolate it from the rest of the network and perform a ‘nested’ NMFIB on it, resulting in a link partition of the subnetwork with two smaller link communities. Subsequently, we decide whether to accept this bipartition by a special method based on the link partition quality. We summarize the algorithm NMFIB using the following recursive algorithm:To mitigate the above problems, we extend our original NMF method proposed above to a more practical one, namely NMFIB, meaning “NMF with iterative bipartition”. In NMFIB, we first divide a network into two link modules using NMF with the number of communities AlgorithmP = NMFIB(G)//G is a network, P is a link partition of G.P = {E(G)};//E(G) denotes the edge set of G.1. G into two link modules N1 and N2 by NMF;2. Divide E(N1)E(N2) = Φ, E(N1)E(N2) = E(N).//P;3. If the link partition quality cannot be improved by this bipartition, return //the quality function is to be introduced later.P1 =  NMFIB(N1);4. P2 =  NMFIB(N2);5. P = P1P2.6. Return G, so as to obtain a superior link community structure. There are several measures for community structures, but most of them are defined for node communities DSubsequently, our remaining task for NMFIB is focused on determining the termination condition for the repetitive process of subdividing the links of network m links and n nodes, P = {P1, P2, …, cP} is a partition of the links into c communities. The number of links in community z, zP, is zm = |zP|. The number of induced nodes, all nodes that those links touch, is zn = |∪eijPz,∈ {i, j}|. The link density zD of zP isFor a network with zm normalized by the minimum and maximum numbers of links among zn connected nodes. Thus, zD  = 1 when zP is a clique, or zD  = 0 when zP is a tree. In particular, we assume that zD  = 0 if zn  = 2 without loss of generality. In essence, zD measures how ‘clique-ish’ versus ‘tree-ish’ zP is. Then, the partition density, D, is the average of zD, weighted by the fraction of links that are present:This is D, the determination of acceptance of the bipartitions for link communities will be independent on the order in which the bipartitioning queue happens in the above iterative procedure. Let we divide an arbitrary community r into two sub-communities r1 and r2. The community structure will become P' = {P1, …, rP-1, rP1, rP2, rP+1, …, cP}, and the its D-value will beIt is worthy of note that, when using function D, will beD-value only depends on this community and its bipartition result, and thus the determination of acceptance of the bipartitions for link communities will be independent on the order of the bipartitioning queue.Then, considering a bipartition for an arbitrary community, the variation of its In order to evaluate the performance of our above method, we tested it both on benchmark synthetic networks and on some widely used real-world networks. The synthetic networks allow us to test the ability of different methods to detect known communities under controlled conditions, while the real networks allow us to observe its performance under practical conditions. Further, we applied our method on a large biological network derived from real world data.DFurthermore, we compared our method with two well-known and highly related methods. The first is a model-based method for link communities proposed by Ball et al All experiments are done on a single Dell Server (Intel(R) Xeon(R) CPU 5130 @ 2.00 GHz 2.00 GHz processor with 4 Gbytes of main memory). The source code of the algorithms used here can all be obtained from the authors. Especially, the code of our methods, which are written as two functions NMF  and NMFIB (our method with iterative bipartition) in Matlab, is available in Recently, several types of synthetic benchmarks have been proposed for node communities n  = 10000 nodes each, divided into two overlapping (link) communities. We placed x nodes in the first community only, i.e., these nodes have connections exclusively within the community, y nodes in the second community only, and the remaining z = n-x-y nodes in both communities, with equal numbers of connections to nodes in these two communities on average. We set the expected degree of all nodes to a fixed value <k>. We also varied the parameters x, y, z, and <k> to generate networks with stark community structures or no structure at all, so as to vary the difficulty of the network instances posed to the algorithms.As done in the experiment designed by Ball et al. in z  = 500, divided the remaining nodes evenly , and varied the value of <k> from 1 to 16 with an increment of 1. For the second set of tests, we again set the overlap at z  = 500 but fixed <k>  = 10 and varied the ratio between x and y. Finally, for the third set of tests, we set <k>  = 10, constrained x and y to be equal, and varied the amount of overlap z.We performed three sets of tests. In the first set of experiments, we fixed the size of the overlap between the communities at priori information for both our NMF method and Ball’s method in this comparison. In As the actual number of communities of the benchmark networks used here is known to be 2, for fairness we make it as a S be the set of truly overlapping nodes and V be the set of predicted overlapping nodes, the Jaccard index is J = |S∩V|/|S∪V|. This index is a standard measure of similarity between sets that rewards accurate identification of the overlap while penalizes both false positives and false negatives. Furthermore, we adopted the Jaccard index to compare the two algorithms’ ability for identifying overlapping (link) communities using the same sets of network instances. Let DD as the quality metric to compare their results. Fortunately, the extended map equation As real-world networks may have some unique topological properties not present in synthetic ones, we now consider some widely used real networks to further evaluate the performance of these algorithms. All the networks we used here are obtained from Newman’s website priori information needed by Ball’s method and our NMF method, and compare these three methods under the condition that the number of communities is the same. The compared results are shown in In the following, in order to evaluate these methods fairly and completely, we perform three sets of tests. First, we use the number of communities attained by Ahn’s method as a Further, we compared the performance of our NMF method with iterative bipartition (NMFIB), Ball’s method with iterative bipartition, as well as Ahn’s method. At this time, all these methods can determine the number of communities automatically. The comparison of these algorithms is shown in c got by the corresponding iterative bipartition method. The compared results are shown in Finally, we compare our NMF method with iterative bipartition  and our original NMF method  with the given number of communities To sum up, our method with iterative bipartition not only has a higher clustering quality compared with other methods, but also it can determine the number of communities automatically. Thus, it may be more suitable for use when detecting link communities on unexplored real networks.Saccharomyces cerevisiaeThe large real network we selected for a particular application is the protein-protein interaction (PPI) network of budding yeast p-value to quantify the significance of the term. To quantify the biological significance of a community structure, we used as quality metric the average number of significantly enriched GO terms with p-values less than a given threshold for all communities. The larger this average number of significant GO terms, the more biologically significant the community structure is.We used the Gene Ontology (GO) terms p-value thresholds tested. It stands as an example of the consistent superior performance of our method over all compared competing methods.Here we compared the results of our method NMFIB, Ball’s method with iterative bipartition and Ahn’s method, since all of them can automatically determine the number of communities. The results are shown in zω’s, to characterize the sizes of different communities, which may enable our model to be more flexible in describing link communities. Then, we fitted the model parameters by taking it as an optimization problem, and used an approach of nonnegative matrix factorization to solve it. Thereafter, we extended the above method by introducing a strategy of iterative bipartition. This leads to a new method, namely NMFIB, that we show to be more suitable for structure discovery in unexplored and large networks. Also, this iterative bipartition process is suitable to be used to improve other model-based methods, such as Ball’s method. We tested our method both on synthetic benchmarks and on real-world networks including an application on a large biological network, and compared it with two well-known and highly related methods. Experimental results demonstrated the superior performance of our method over the competing methods in the detection of link communities.In this work, we proposed a generative model for link communities based on Ball’s model izX (elements in the auxiliary nonnegative matrix X) that falls below a predetermined threshold, and improve the efficiency of our NMF method by using a technique of sparse matrix calculations. This is one of the directions for our future work. Moreover, in the current work, we only used partition density DOur model was mainly designed to describe link communities, but it can be also extended to find node communities. A simple and approximate method is assigning each node solely to the community to which it most strongly belongs in the overlapping (link) community structure. But in the future, we wish to improve our model from some other viewpoints, and make it able to describe link communities as well as node communities, naturally and in a similar way, rather than being a simple extension. Furthermore, as discussed, the efficiency of our method is competitive with that of Ball’s method, and it will become more efficient when introducing the strategy of iterative bipartition. Nevertheless, in order to deal with some very large networks such as the WWW, the Internet, etc, its efficiency needs still to be improved. Possibly, we can devise a pruning strategy that sets to zero any


Pulmonary hypertension (PH) represents an important complication of idiopathic pulmonary fibrosis (IPF) with a negative impact on patient survival. Herpes viruses are thought to play an etiological role in the development and/or progression of IPF. The influence of viruses on PH associated with IPF is unknown. We aimed to investigate the influence of viruses in IPF patients focusing on aspects related to PH. A laboratory mouse model of gamma-herpesvirus (MHV-68) induced pulmonary fibrosis was also assessed.Lung tissue samples from 55 IPF patients and 41 controls were studied by molecular analysis to detect various viral genomes. Viral molecular data obtained were correlated with mean pulmonary arterial pressure (mPAP) and arterial remodelling. Different clinical and morphological variables were studied by univariate and multivariate analyses at time of transplant and in the early post-transplant period. The same lung tissue analyses were performed in MHV-68 infected mice.p = 0.0003) and only herpes virus genomes were detected. Viral cases showed higher mPAP (p = 0.01), poorer performance in the six minute walking test  and higher frequency of primary graft (PGD) dysfunction after lung transplant (p = 0.02). Increased arterial thickening, particularly of the intimal layer (p = 0.002 and p = 0.004) and higher TGF-β expression (p = 0.002) were demonstrated in viral cases. The remodelled vessels showed increased vessel cell proliferation (Ki-67 positive cells) in the proximity to metaplastic epithelial cells and macrophages. Viral infection was associated with higher mPAP (p = 0.03), poorer performance in the 6MWT (p = 0.008) and PGD (p = 0.02) after adjusting for other covariates/intermediate factors. In MHV-68 infected mice, morphological features were similar to those of patients.A higher frequency of virus positive cases was found in IPF patients than in controls (Herpesviral infections may contribute to the development of PH in IPF patients. Idiopathic pulmonary fibrosis (IPF), morphologically usual interstitial pneumonia, is a chronic progressive disease of unknown aetiology with irreversible scarring in the lung Recent studies have shown that PH measured with right-heart catheterization or transthoracic echocardiography in IPF patients is associated with low DLCO, shorter walk distances, and desaturation during exercise However to date no clinical or biohumoral parameters are available as predictive markers of PH in IPF patients. Several authors have shown that PH in IPF relates poorly to the degree of pulmonary function test and is not always confined to advanced disease Pathogenic mechanisms of IPF and of its main complications are complex and still largely unknown although different hypotheses have been proposed, varying from previous chronic inflammation with subsequent widespread fibrosis to abnormal wound healing. Increasing evidence suggests that a key event in IPF is deregulated epithelial cell function with ongoing alveolar epithelial injury and an associated abnormal host repair response, leading to patchy and heterogeneous morphological changes Alveolar epithelial cells, one of the most important targets of the virus, are then involved in the activation of different signalling pathways responsible for fibrotic lung remodelling. In viral animal and cell models several profibrogenic cytokines/chemokines have been shown to be over-expressed and among these transforming growth factor beta 1 (TGF-β1) plays a key role The aim of the study was to investigate the relevance of respiratory viruses in IPF patients, with special emphasis on cases complicated by PH. For this purpose a large number of IPF patients and control subjects including other non-IPF diffuse parenchymal lung diseases (DPLDs) were studied. Viral molecular data were correlated with mean pulmonary arterial pressure (mPAP) and arterial remodelling. Different clinical and morphological variables were studied by univariate and multivariate analysis at time of transplant and in the early post-transplant period.A mouse model was used to examine whether murine gammaherpesvirus-68 (MHV-68), genetically and biologically similar to EBV, contributes to the development of vessel remodelling other than pulmonary fibrosis.Native lungs from 55 IPF patients who underwent lung transplantation (LT) between September 1998 and February 2010 in our Centre were studied . The control group consisted of 22 native lungs from other non-IPF -DPLDs-  and 19 normal lungs (13 non implanted donor lungs and 6 autopsy cases). Prior to LT after the diagnosis of IPF, the majority received oral prednisone while a minority were treated with prednisone and azathioprine.Each patient underwent pulmonary function testing, high resolution computed tomography and right heart catheterization. These tests were performed in all patients at the time of waiting list inclusion and before LT. For the present study clinical/functional parameters collected at the time of LT were considered.The diagnosis of interstitial lung disease was based on the diagnostic criteria of the American Thoracic Society/European Respiratory Society Consensus Classification System Moreover, short term post-transplant follow-up (12 months after LT) was also considered in all IPF cases, mainly focusing on primary graft dysfunction (PGD) and acute rejection, both considered causes of early and late graft dysfunction. PGD and acute rejection were graded as proposed by the International Society for Heart and Lung Transplantation (ISHLT) Working Group Written informed consent was obtained from all subjects. The work was approved by the Institutional Ethics Committee. Relevant clinical data of the IPF and non-IPF DPLD population are summarized in Nucleic acids were extracted from fresh lung tissues sampled from different areas (∼1 mg of tissue) of all 96 cases (55 IPF patients and 41 control subjects) using a modified RNAzol method Samples were considered as true positive when the reproducibility of PCR analysis was verified a second time. Amplicon specificity was verified by direct cycle sequencing as previously described Since EBV was identified with high frequency in IPF patients, RNA-in situ hybridization-ISH  was performed to identify the viral target cells. To quantify DNA viral genome copies real time PCR was also carried out in EBV positive cases whose additional DNA was available using artus® EBV TM PCR KIT The extent of fibrosis was evaluated in Masson’s trichrome stained sections analyzing 10 random fields using computer-assisted morphometric software (Image ProPlus® 5.1), as previously reported Arterial thickening was quantified using computer-assisted morphometric software on elastic-Van Gieson (EVG) stained lung sections focusing on muscular arteries of an average diameter of 300 µm (range: 100–500 µm). In particular, medial thickening (MT) was evaluated as previously described in at least 5 arterial sections. The same approach was used to measure intimal thickening (IT): MT% =  × 100; IT% =  × 100 For Ki-67 immunohistochemistry, after microwave antigen retrieval, the sections were treated with normal serum  and incubated for 60 min with the primary monoclonal antibody anti-Ki67  at a dilution of 1∶100. Sections were subsequently incubated with rabbit HRP polymer  for 30 min. Immunoreactivity was visualized with 3-3′-diaminobenzidine (Dako). Negative controls processed omitting the primary antibody did not show any reaction. Ki-67 expression and TUNEL positivity were evaluated only in blood vessel cell components. All evaluations were performed blindly.5 PFU MHV-68 and euthanized 7 days (n = 3), 14 days (n = 6) and 23 days (n = 3) post infection (post infection). Uninfected mice (n = 4) served as controls.Female, 5 to 8 week old CD-1 mice [Hsd:ICR (CD1)] were purchased from Harlan Laboratories, UK and housed at the University of Liverpool under specific pathogen-free conditions. Mice were intranasally infected with 4×10Immediately after death, lungs were collected and parts frozen at −80°C for RNA extraction: others were fixed in 10% buffered formalin and routinely embedded in paraffin wax. All experiments were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of The National Institutes of Health. The protocol was approved by UK Home Office regulations and under Project Licence number 40/2483 and Personal Licence number 60/6501. All infections were performed under light isoflorane anaesthesia. All experiments were carried out in manner to minimize suffering.Consecutive paraffin-embedded sections were stained with haematoxylin and eosin for histology, with Masson’s trichrome stain for fibrosis extension and with EVG for vessel remodelling, both morphometrically measured as above. The evaluation of arterial thickening focused on pulmonary arteries with an average diameter of 82 µm ranging from 43 to 100 µm. The assessment of vascular remodelling also included an evaluation of apoptosis and proliferation of vessel cell components using the TUNEL technique and Ki-67 immunostaining, as described above. Ki-67 antibody used for this analysis was rabbit polyclonal . Immunohistochemistry was also employed for the detection of MHV-68 antigen, to highlight lung macrophages (expression of lysozyme) and to demonstrate TGF-β expression . Viral tRNA was demonstrated by RNA-ISH Data were analyzed using the SAS statistical software version 9.1.3 . For quantitative variables the results are expressed as mean values ± standard deviation if normally distributed, otherwise as median, Q1–Q3. The normality of distribution of quantitative variables was tested by means of Shapiro-Wilk statistics. For quantitative characteristics, differences between subjects with and without viral infection were evaluated by using the Mann-Whitney test. The prevalence of specific conditions was expressed as a percentage, and differences between groups were evaluated using the χ-squared test and exact Fisher’s test, as adequate.Unadjusted and adjusted relationships between virus and clinical/morphological features were tested with general linear models (GLM procedure). In all analyses, a two-tail level for significance was set at 0.05. Simple logistic regression models were applied to estimate odds ratios (ORs) for PGD using recipients’ and donors’ characteristics as predictors. A multivariate logistic model was used to estimate the adjusted OR of virus for PGD, controlling for PAP>25 mmHg. Considering that incidence was quite common in the study population, adjusted risk ratios (RRs) were approximated from adjusted ORs p = 0.0003). Normal lungs were all negative. Herpes viruses were the only detected genomes in IPF and EBV resulted as the most frequent, present in more than half of IPF patients. In 2 cases double infection was found. EBV was never detected in control cases . Gene sequencing of all amplicons showed a high homology (from 95% to 99%) with human viral genome sequences. RNA-ISH for EBV (EBER) identified viral RNA in 40% of EBV-PCR positive IPF cases. The positivity was detected in alveolar epithelial cells other than within monocytes/macrophages . Normal lungs did not show arterial remodelling  and metaplastic alveolar epithelial cells . Median TGF-β scores in the alveolar epithelium were significantly higher in IPF patients than in the DPLD group . In normal lungs, TGF-β expression was restricted to only scattered intraalveolar macrophages. Arterial remodelling in IPF cases showed a median total thickness score of 43.8% (Q1–Q3: 35.7–53.1%) with IT and MT scores of 17.7% (Q1–Q3: 13.5–24%) and 26.2% (Q1–Q3: 21.8–28.9%) respectively. These values were significantly lower in the DPLD group  and worse performance in the 6MWT  than virus negative cases  than virus negative cases. The intimal layer was most severely affected  (p = 0.002)   A,B,C,D.= 0.004)  while ce= 0.004)  A and B.= 0.002)  A,B,C,D.p = 0.03) after adjusting for other related covariates and intermediate factors . Moreover, viral presence was an independent marker of significantly poorer performance in the 6MWT (p = 0.008) using the GLM adjusting for covariates . When we considered the short-term post-transplant follow-up we found a higher frequency of PGD  and, although not statistically significant, an increased rate of acute rejection  in virus positive compared to negative IPF cases. A multivariate analysis identified an increased risk for PGD to be associated with viral infection independently of the major recipient/donor characteristics that are usually considered to influence PGD, including PH   .Analysis of MHV-68 DNA load showed, as previously demonstrated, that viral infection peaked on day 7 post infection The histological examination of uninfected control animals did not identify any pathological changes. On day 7 post infection, all infected animals exhibited increased, macrophage-dominated interstitial cellularity, mild type II pneumocyte hyperplasia and perivascular inflammatory infiltration. The latter infiltrates were associated with slight patchy collagen deposition. Scattered individual macrophages and metaplastic alveolar epithelial cells were found to express MHV-68 antigen. Viral latency, represented by the expression of viral tRNA, was detected in type II pneumocytes and alveolar macrophages. On day 14 post infection, mild multifocal, almost diffuse fibrosis of alveolar septa was observed with a median of 26% (range: 17–38%). In some animals, this was associated with random patches of collagen deposition. Numerous macrophages were seen within the interstitium and often around arteries. In addition, some type II pneumocytes exhibited TGF-β expression  A, B.Arteries were assessed for vessel remodelling and compared for the thickening scores. In uninfected control mice, the average score was 16.5. Seven and 14 days post infection, it was 18 and 24.5 respectively  A,B,C,D.In the present study different respiratory viruses were investigated in lungs from a large cohort of IPF patients and the control group  displaying the prevalent role of herpes viruses. The work highlights the broad impact of herpesviral infection, particularly EBV, in the disease emphasizing its association with a more severe disease phenotype as IPF with associated higher mPAP and worse 6MWT. A multiple logistic regression analysis demonstrated that herpesviral infection was independently associated with more marked vessel remodelling, higher value of PAP and worse performance in the 6MWT.Neither clinical data  nor morphological changes (fibrosis extension) had more significant impact than viral infection. Of particular note is that the presence of viral infection had also early-term negative influence on the graft function. IPF virus positive recipients showed a higher frequency of PGD, independent of other recipient and donor characteristics. Viral products and/or the activated immune system related to viral infection in native IPF lungs may potentiate the immune response to alloantigens after transplantation, thus resulting in early graft dysfunction. Only one previous study performed on 24 IPF patients reported more rapid disease progression in EBV positive cases. The majority of viral cases died from respiratory failure at a mean of 41 month follow up. In the study PH was not specifically considered The reported frequency of herpes viruses in IPF lungs ranges from 30 to 100%. These differences may be related to technical sensitivity, disease heterogeneity or selection of patients. The presence of herpes viruses detected in our cases may even be underestimated due the fact that the molecular investigation was performed on tissue samples from end-stage diseases. Viral clearance, after initial injury, could occur in some cases with persistence and progression of lung injury due to activation of an immunological response, as it occurs in other chronic virus-related diseases, e.g. dilated-cardiomyopathy-post-myocarditis To the best of our knowledge no attempt has been made to specifically investigate the influence of herpes viruses on arterial remodelling and PH in IPF patients.Several types of viruses, particularly those of the herpes family, have been found to be associated with vessel remodelling, development of atherosclerosis and clinical features of hypertension vivo has not been well defined, in vitro and experimental studies have demonstrated an important influence of this cytokine in muscle/fibroblast proliferation, endothelial-mesenchymal transition, and extracellular matrix production of intimal and medial layers Although the function of TGF in vascular cell growth in The demonstration of a direct causal relationship between herpesvirus infection and vessel remodelling/PH in IPF would require longitudinal studies of the same patients, an impossible task with lung tissue but attainable with bronchoalveolar lavage or peripheral blood samples. However this limitation has been partially overcome in the present study using a laboratory MHV-68 infected mouse model. Indeed, in these animals 2 weeks after infection significant arterial remodelling and increased TGF-β expression was seen, as those observed in clinical lung specimens from IPF patients with high mPAP.In summary, our results demonstrated for the first time a different phenotype of virus-positive IPF patients. In particular virus-positive IPF cases showed more pronounced vessel remodelling and a higher mPAP and significantly higher PGD after transplantation. While there is large mechanistic evidence of epithelial herpesvirus-associated alveolar injury, the effect of these viruses on the pulmonary vasculature in IPF merits investigation. A deeper knowledge of viral-induced pathways in endothelial cells could give new insights for a targeted therapeutic approach of this important complication in the subgroup of patients (virus positive cases). In this context, the high degree of similarity between MHV-68 infection of CD-1 mice and virus positive IPF indicates that this is an excellent model with which to study pathogenesis and interventions.Figure S1Cell apoptosis in IPF lung tissue. Endothelial cell apoptosis (TUNEL positive) well seen in a capillary surrounded by extensive fibrosis (arrows). Bar scale: 10 µm.(TIF)Click here for additional data file.Figure S2Cell apoptosis in MHV-68 infected CD1 mice lungs. Endothelial cell apoptosis  well seen in a capillary of high remodeled area. Note apoptotic body inside a macrophage (arrow head). Bar scale: 10 µm.(TIF)Click here for additional data file.Table S1Virus-positive vs virus-negative IPF .(DOC)Click here for additional data file.Table S2Unadjusted relative risks  for post-transplant PGD – recipients and donors characteristics used as predictors.(DOC)Click here for additional data file.


According to this process, a distance must be maintained between the plasma initial stage and the deposition stage to allow the plasma to diffuse to the substrate. Raman spectra revealed that a higher hydrogen concentration promoted the synthesis of a high-quality graphene film. The results demonstrate that plasma-assisted thermal CVD is a low-cost and effective way to synthesis high-quality graphene films at low temperature for graphene-based applications.Plasma-assisted thermal chemical vapor deposition (CVD) was carried out to synthesize high-quality graphene film at a low temperature of 600°C. Monolayer graphene films were thus synthesized on Cu foil using various ratios of hydrogen and methane in a gaseous mixture. The  The carriers in graphene are transported in the π-orbitals that are perpendicular to the surface so the optical transparency of a single layer of graphene can be as high as approximately 97%, and it can exhibit excellent electronic properties with reported mobilities of between 3,000 and 27,000 cm2/V·s [Graphene, a sp cm2/V·s -3. Vario cm2/V·s -7. Chemi cm2/V·s . Sulfuri cm2/V·s . This me cm2/V·s ,12. The  cm2/V·s -19. This cm2/V·s , and sur cm2/V·s ,22. Howe2) flow rates from 5 to 20 sccm at a temperature of as low as 600°C. Figure Throughout the experiments, plasma-assisted thermal CVD was used to synthesize graphene films on polycrystalline copper foils with various hydrogen  gas, flowing at 1 sccm, was the carbon source; it was mixed with various flows of H2 and fed into the tube for 5 min to form a monolayer of graphene. Subsequently, the sample was rapidly cooled by removing it from the hot zone of the thermal furnace. The synthesized graphene films were transferred onto the SiO2 (300 nm)/Si substrates by etching away the copper foil in an iron chloride (FeCl3) solution. Prior to wet etching, a 200-nm-thick thin film of PMMA (poly-methyl methacrylate) was spin-coated on the top of graphene/copper foil and then baking it at 130°C for 1 min. The PMMA/graphene thin films were washed with dilute hydrochloric acid solution to remove the metal ions and then rinsed in DI water. PMMA/graphene films were placed on the SiO2 (300 nm)/Si substrate, and the PMMA was then dissolved in an acetone bath over 24 h.Graphene films were grown on a 25-μm-thick copper foil  using the proposed plasma-assisted thermal CVD system by a method similar to one described elsewhere . Prior t4/H2 mixed plasma and CHx radicals. The gaseous CHx radicals recombined with each other after they had floated for a certain distance, and the metastable carbon atoms and molecules formed a sp2 structure on the copper surface. Most importantly, the most effective length for growing graphene between the plasma and the center of the hot zone was approximately 30 cm herein.Figure 4/H2 mixed gas with various proportions of H2[n = 1, 2, 3,…) [n > 1) in a strong electric field, ionizing the hydrogen atom as the electrons were excited to high energy levels. The transition from n = 3 to n = 2 is called H-alpha (Hα) and that from n = 4 to n = 2 is called H-beta (Hβ) with emitted wavelengths of approximately 656 and 486 nm, respectively. After ionization, the excited electron recombined with a proton to form a new hydrogen atom, yielding the Hx spectra. In this case, the ionized gas of CH4/H2 recombined as CHx radicals moved after a certain distance. Figure 2 flow rates and a gas pressure of 0.5 Torr. In this work, the recombination lines of the atomic  and molecular (H2 = 550 to 650 nm) hydrogen dominate the emission spectra. The emission spectra of CH radical (430 nm) and C2 dimers (541 nm) after the introduction of CH4 are obtained [α occurs when hydrogen is ionized where intensity increases with the H2 flow rate. The intensity of the CH spectral peak declined slightly as the H2 flow rate increased, revealing that increasing the H2 concentration improves the rate of decomposition of the mixture gas. The C2 dimers in plasma during the plasma-assisted thermal CVD are critical to the formation of various carbon materials [Figure ons of H2. Accordi 2, 3,…) ; electroobtained . Hα occuaterials . Further2 flow rates from 5 to 20 sccm at a low temperature of 600°C. Typical features of the monolayer graphene are observed. They include (1) a 0.5-G-to-2D intensity ratio and (2) a symmetric 2D band that is centered at approximately 2,680 cm−1 with a full width at half maximum (FWHM) of approximately 33 cm−1. The 2D band is related to the inter-valley double resonant Raman scattering, and the peak of the G band is produced by the Eg2 phonon at the center of the Brillouin zone around approximately 1,580 cm−1. The D band is associated with the breathing modes of the sp2 atoms and is activated by a defect. Sharp single Lorentzian 2D band was observed at approximately 2,700 cm−1 when the H2 flow rate exceeded 10 sccm. The intensity of the D band decreased with increasing H2 flow rate indicating not only increased crystallization of graphene but also in CVD graphene on copper, the formation of C-H bonds as out-of-plane defects. Overall, hydrogen plays an important role in the growth of graphene and in determining its quality. This result is consistent with Figure Figure 2 flow rate increases, Id/Ig declines from 0.33 to 0.13 and I2d/Ig increases from 0.98 to 2.29. The lower 2D band and higher D band reveal that the more disordered graphene growth, the lower is the H2 flow rate. Interestingly, the 2D-peak FWHMs (39 to 35 cm−1) of the series of samples varied slightly with the H2 flow rate because the low solubility of carbon in copper makes graphene growth self-limiting, and a higher H2 concentration improves the inter-valley double resonance in the Raman spectrum.Figure 2 flow rates. When the H2 flow rate increased, the Raman spectra of the samples have I2d/Ig ratios that increase from 0.98 to 2.29 and the FWHMs of the 2D band that decrease from 39 to 35, both indicate that the graphene film is high quality. Plasma-assisted thermal CVD is a more effective method for depositing high-quality graphene films on metal substrates.This study elucidates the effect of hydrogen on graphene grown on Cu by plasma-assisted thermal CVD at a low temperature of 600°C. The mechanism of growth of graphene by plasma-assisted thermal CVD was clarified by obtaining plasma emission spectra at various HThe authors declare that they have no competing interests.SHC (Chan) designed the study and wrote the paper. WTL and MCL analyzed the data. SHC (Chen), YCL, and CCK are advisors. All authors read and approved the final manuscript.


A new crystal-mounting method has been developed that involves a combination of controlled humid air and polymer glue for crystal coating. This method is particularly useful when applied to fragile protein crystals that are known to be sensitive to subtle changes in their physicochemical environment. Protein crystals are fragile, and it is sometimes difficult to find conditions suitable for handling and cryocooling the crystals before conducting X-ray diffraction experiments. To overcome this issue, a protein crystal-mounting method has been developed that involves a water-soluble polymer and controlled humid air that can adjust the moisture content of a mounted crystal. By coating crystals with polymer glue and exposing them to controlled humid air, the crystals were stable at room temperature and were cryocooled under optimized humidity. Moreover, the glue-coated crystals reproducibly showed gradual transformations of their lattice constants in response to a change in humidity; thus, using this method, a series of isomorphous crystals can be prepared. This technique is valuable when working on fragile protein crystals, including membrane proteins, and will also be useful for multi-crystal data collection. This substance is widely used not only as a stationery glue and as a laundry starch but also in biological applications: for instance, as a cryopreservation reagent for embryos  and 12%(w/w)], PVA3500 [13%(w/w)] and PVA2400 [15%(w/w)] were dissolved in water by brief heating (363 K) and stirring.As the water-soluble polymer, we used polyvinyl alcohol (PVA) with average polymerization degrees ranging from 2000 to 4500 . PVA2000, PVA2400 and PVA4500 were supplied by Japan VAM & POVAL Co. Ltd, Japan . PVA3500 was purchased from Wako Pure Chemical Industries Ltd (product No. 163-16355). Aqueous solutions of PVA4500 [8%(w/w) and 13%(w/w) PVA4500 gave a powder diffraction pattern of hexagonal ice after flash-cooling without any humidity control. Similarly, glue exposed to air of 92.3% RH (relative humidity) produced a powder diffraction pattern of cubic ice , but at 92.0% RH no ice ring was observed . Since the final resultant concentrations of PVA are affected by vapour pressure, i.e. atmospheric humidity  PVA4500 was used as the mounting glue in all of the experiments if not otherwise specified.For flash-cooling experiments, humidity conditions that vitrify the PVA solutions themselves were examined by X-ray scattering. Initial solutions of 8%. Dry air was supplied by an N2-gas generator  and an HOHMI YC-4R air compressor . Saturated-humidity air was produced by bubbling the dry air in a water bath kept at 301 K and cooling to room temperature (296 ± 1 K with a temperature stability of ±0.3 K h−1) during transportation. Next, it was mixed with the dry air and the total flow rate was brought to 3 l min−1 or greater using two SEC-N series digital mass-flow controllers  controlled by a PC running Windows 7. Although this handmade humidity generator was used in this study, we confirmed that a modified version of the commercial humidity generator HUM-1 , which can produce humid air at a high flow rate, also worked for this purpose. The humidity-control gas was transported to a humid air nozzle, which was produced from a flexible Teflon tube with an inner diameter of 10 mm. The outlet temperature and the relative humidity were measured inside the humid air nozzle using an SHT71 humidity sensor . This humidity-control device can control the humidity range between 0.1 and 94.5% RH. Since the humidity was regulated by the mixture of gases, humidity change took place at a rate of between 0.2 and 1% RH min−1.We developed a simple humidity-control apparatus equipped with a two-flow humidity generator Fig. 2a. Dry a2.3.2.3.1.−1). The humid air nozzle of the apparatus was placed under the sample  and the humidity was adjusted; typically, the initial humidity was 83% RH. The cryostream nozzle was removed from the sample position to avoid interference with the humid air . In the following steps performed at room temperature, the intensity of X-ray radiation should be minimized in order to reduce radiation damage. In the case of our beamline, SPring-8 BL38B1, an aluminium plate with a thickness of 600–1000 µm was used to attenuate the X-ray beam with the full photon flux of 1.17 × 1011 photons s−1 to 1/10 to 1/40 at a wavelength of 1 Å, respectively.The humidity-control apparatus was installed on the SPring-8 BL38B1 beamline equipped with a one-axis rotation goniometer , ADSC Quantum 210 or 315 CCD detectors and a GN2 nitrogen cryostream system (Rigaku Co.). All of the following operations were performed at room temperature  PVA4500 was used except for certain crystallization conditions. PVA interacts with ions, especially multivalent anions such as tartrate, phosphate and sulfate ions, and forms a physical hydrogel. Therefore, it is often difficult to thoroughly coat the crystals owing to a varying degree of hydrogel elasticity that depends on the concentration and the valences of the ions. It is known that plasticizing reagents such as glycerol, ethylene glycol and polyethylene glycol 400 prevent gel formation and that 5%(v/v) glycerol is sufficient to reduce gel formation  glycerol before transferring the crystal into the glue. (ii) Glycerol was added to the crystallization solutions to a final concentration of 5%(v/v). (iii) In the case of crystallization solutions containing a high concentration of multivalent ions (e.g. greater than 1 M), the glue was prepared with equal volumes of pure glycerol and the aqueous PVA solution.An aqueous glue solution consisting of 8% with a typical diameter larger than the crystal . A crystal was directly picked up from a crystallization droplet by the glue-coated loop without removal of the mother liquor surrounding the crystal . The scooped crystal was kept steady for a few seconds so that it could be thoroughly coated and covered by the glue on the loop . Next, the loop was mounted on the diffractometer and exposed to the humid air. This operation should be completed within 10–20 s to minimize transpiration of the crystalline moisture.A small amount of the glue solution was applied to and spread over a crystal-mounting loop (2.3.4.c). To avoid this, a suitable humidity level needs to be established immediately. The swelling of the glue and consequent formation of crystalline ice at cryogenic temperature is another concern. The protocol for coping with these problems is described in Fig. 3A destructive hydration shock could be induced if the glue were diluted by absorbing a large amount of the droplet solution in the crystal pickup Fig. 2c. To avHKL-2000 suite .Once the humidity had been optimized, it was kept constant without any further change. Before proceeding to the next step, the crystal was maintained at the optimum humidity for 15 min to complete water equilibration. Since lower humidity was preferred in many crystals, the glue was dehydrated and shrunk at optimum humidity . Lysozyme crystals were grown using hanging-drop vapour diffusion at 293 K. Hanging drops were prepared by mixing 5 µl of 35 mg ml2.4.2.Bacillus subtilis was expressed and purified as described previously , which was the lowest concentration to vitrify the mother liquor. However, the crystals were mechanically very fragile and sensitive to environmental changes. Indeed, the crystals gradually delaminated and crumbled once the cover slides of the hanging drops were opened.The bacterial hydrolase RsbQ from 2.4.3.et al.  glycerol.Bovine insulin was obtained from Sigma–Aldrich Co. (product No. I550) and crystallized based on the method reported by Gursky  al. 1992. The cry2.4.4.Blastochloris viridis photoreaction centre (BvRC) crystal was provided by Dr Takayuki Odahara. The crystals of BvRC were grown using sitting-drop vapour diffusion at 288 K with a small glass vessel for protein solution installed in a sealed plastic container. Sitting drops were prepared using 400 µl of 18 mg ml−1BvRC solution in 2.5%(w/v) triethylamine phosphate, 2.5%(w/v) 1,2,3-hexanetriol, 0.3% lauryl dimethylamine oxide (LDAO), 1.51 M ammonium sulfate, 60 mM sodium phosphate buffer pH 7.0. The volume of reservoir solution consisting of 5%(w/v) triethylamine phosphate, 1.89 M ammonium sulfate, 100 mM sodium phosphate buffer pH 7.0 was 12 ml in each setup. The glue for the HAG mount was a mixture prepared from equal volumes of pure glycerol and 8%(w/w) PVA4500 solution.The bacterial membrane protein 2.4.5.4 synthase (LTC4S) was provided by Dr Hideo Ago  n-dodecyl-β-d-maltoside (DDM) with an equal volume of reservoir solution consisting of 0.04%(w/v) DDM, 5 mM glutathione, 1.6–1.7 M ammonium sulfate, 0.5–0.8 M magnesium chloride, 20 mM MES–NaOH buffer pH 6.5. The crystals of LTC4S were transferred overnight into harvesting solution consisting of 2.4 M ammonium sulfate, 50 mM glutathione, 20 mM MES–NaOH buffer pH 6.5 . By employing an HAG mount using 13%(w/w) PVA3500 glue without any other cryoprotectants and an optimized humidity of 69.1% RH, cryoprotection of the RsbQ crystals was successfully achieved and a full diffraction data set was collected  or higher as a permeable cryoprotectant in the conventional cryocooling method, whereas they could be mounted and cryocooled with only 5%(v/v) glycerol in the HAG method. In this case the crystal was first soaked in reservoir solution supplemented with 5%(v/v) glycerol to replace the mother liquor and was then used in HAG mounting. The addition of only 5%(v/v) glycerol was sufficient to prevent gel formation by the 0.4 M phosphate ion contained in the mother liquor. By contrast, for both BvRC and LTC4S crystals the addition of 5%(v/v) glycerol was insufficient to prevent gel formation because both of the crystallization conditions contained high concentrations of sulfate ions (1.9–2.5 M). However, addition of glycerol to the polymer glue was effective for both crystals. The glue prepared by mixing equal volumes of glycerol and 8%(w/w) PVA4500, i.e. a final concentration of 50%(v/v) glycerol and 4%(w/w) PVA, was successfully subjected to cryocooling without ice formation  glycerol was damaged in only 2 min . During the dehydration process to 68.3% RH , a 0.73% shrinkage of the crystallographic a axis and a 1.45% stretching of the c axis were simultaneously observed. With further dehydration from 68.3 to 67.6% RH the crystal suddenly cracked and the mosaicity surged . Rehydration with increased humidity did not lead to recovery of the crystal from cracking.In addition to the cryoprotection effects, we also investigated the transformation of lysozyme crystal lattices in response to humidity changes. Firstly, we estimated the humidity ranges in which glue-coated crystals remain stable. Tetragonal crystals of hen egg-white lysozyme were mounted by the HAG method without any additional cryoprotectants and the humidity was reduced from 87.7% RH to the catastrophic point at which the crystal was damaged . The dehydration process gradually led to lattice transformation along the a and c axes to 78.51 and 38.35 Å, respectively. Moreover, during the following rehydration process the lattice parameters inversely traced the transitions that were observed in the dehydration process. This showed a linear relationship between the humidity and the lattice constants, although a small nonlinear response and increased mosaicity were observed in the first dehydration steps. Such a nonlinear response was also observed by exposing a crystal to constant humidity . In particular, the samples exposed to high humidity (87.3–88.6% RH) showed higher mosaicity (0.55–0.66°) than the others (<0.46°). In terms of the resolution, the data set with the highest resolution was obtained from the 82.7% RH condition, which is in the middle of the tested humidity range (Table 2i.e. a solution of only PVA treated with humidity above 92% RH gave ice-ring diffraction on cryocooling. It has been reported that the water vapour pressure of aqueous PVA (molecular mass 88 kDa) at concentrations of about 60% or below remains approximately at the saturated pressure of water vapour at 304.15 K  by exposure to unsaturated humid air, leading to successful vitrification.The effect of the HAG mount for cryocooling was examined using five lysozyme crystals, each with a different humidity condition, in which no ice-ring patterns were observed but for which the mosaicities increased after cryocooling Table 2b. In pae Table 2. Both ex4.−1) compared with that of the crystallization solution of lysozyme .(i) The nature of retaining moisture and buffering moisture exchange. The glue prevents direct exchange of water vapour between the crystal and the atmosphere. Instead, the glue releases its own moisture, rather than that of the crystal, to the dry atmosphere during crystal handling or humidity changes. This moderate moisture-exchange system protects fragile crystals from a hydration or dehydration shock. In addition, the hypotonic property of the polymer solution retains the moisture of a crystal. The osmotic pressure of the glue solution depends on the concentration of its solute and is also affected by the polymerization degree of the solute polymer according to the Flory–Huggins theory; a longer polymer gives a lower osmotic pressure at a given vapour pressure. Indeed, the osmolality of the glue is almost negligible  High viscosity and slow diffusion. The high viscosity of the glue guards the crystals from mechanical stress and slows the diffusion of solvents, solutes and PVA itself. The high viscosity may retard changes in the chemical environment surrounding a crystal. The effects might also mitigate osmotic shocks (Sugiyama (iii) Affinity for crystallization solutes. The glue might provide a similar chemical condition to the crystallization drop by absorbing the crystallization solution during crystal pickup. Even in dried conditions, the absorbed chemicals might be trapped by the glue through interaction with the hydroxyl groups of PVA, preventing the condensation of chemicals inside the crystal.(iv) Suppression of ice crystals. Similarly to permeable cryoprotectants, the hydrophilic polymer suppresses the formation of ice crystals in the solution around the crystals. Moreover, the hydrophilic glue can absorb the excess crystallization solvent that usually contributes to ice formation. In this sense, the use of the polymer glue is different from oil coating, in which case a small amount of the excess solvent may be left at the crystal–oil border.Our results demonstrate the high feasibility of the HAG method for crystal mounting and cryocooling. We speculate upon several possible reasons for the feasibility and efficacy of this method.c). However, glue-coated lysozyme crystals showed a different response to that of the uncoated crystals to humidity changes . In the hypertonic condition, the polymer concentration exponentially correlates with the osmotic pressure. In addition to the pressure, mechanical force induced by dehydration of the PVA hydrogel and salt displacement might cause significant stress to crystals. In some cases, the glue surface became lumpy and salt crystals appeared during the dehydration processes. The surface roughness of the glue might reflect the formation of PVA hydrogel, in which physical networks of polymer molecules are formed by hydrogen bonds and ionic interactions together with polar or ionic solutes. To avoid unrecoverable damage, it might be necessary to optimize the humidity and the amount of the plasticizing reagents.Meanwhile, the mechanism that causes the catastrophic damage upon dehydration remains unclear Fig. 5a. In thet al., 2012et al., 2012The controllability of the osmotic pressure will aid crystallographers to manage lattice constants and isomorphism when dealing with a series of crystals at room temperature. This feature might be useful for multi-crystal data collection using synchrotrons or X-ray free-electron lasers (Kern 5.The new crystal-mounting method, the HAG method, is an effective way of handling fragile protein crystals and is compatible with experiments at both room and cryogenic temperatures. Furthermore, treatment of samples with proper humidity could produce desired lattice constants, potentially resulting in improved isomorphism for multi-crystal data collection using a series of crystals. This feature is required for experiments with radiation-sensitive crystals or when conducting experiments at room temperature using a synchrotron or an X-ray free-electron laser X-ray source. We are in the process of further improvement of the method.


Danio rerio) embryos with T-box transcription factor 5 (tbx5) deficiencies, mimicking human Holt-Oram syndrome.Dysmorphogenesis and multiple organ defects are well known in zebrafish  antisense RNA and mismatched antisense RNA in the 1-cell stage served as controls, while zebrafish embryos co-injected with exogenous growth hormone (GH) concomitant with tbx5-MO comprised the treatment group.Using an oligonucleotide-based microarray analysis to study the expression of special genes in tbx5-MO knockdown embryos were quantified and observed at 24, 30, 48, 72, and 96 h post-fertilization. Though the understanding of mechanisms involving GH in the tbx5 functioning complex is limited, exogenous GH supplied to tbx5 knockdown zebrafish embryos is able to enhance the expression of downstream mediators in the GH and insulin-like growth factor (IGF)-1 pathway, including igf1, ghra, and ghrb, and signal transductors , and eventually to correct dysmorphogenesis in various organs including the heart and pectoral fins. Supplementary GH also reduced apoptosis as determined by a TUNEL assay and decreased the expression of apoptosis-related genes and proteins (bcl2 and bad) according to semiquantitative reverse-transcription polymerase chain reaction and immunohistochemical analysis, respectively, as well as improving cell cycle-related genes (p27 and cdk2) and cardiomyogenetic genes .The attenuating effects of GH in tbx5 knockdown causes a pseudo GH deficiency in zebrafish during early embryonic stages, and supplementation of exogenous GH can partially restore dysmorphogenesis, apoptosis, cell growth inhibition, and abnormal cardiomyogenesis in tbx5 knockdown zebrafish in a paracrine manner.Based on our results,  PCR conditions were comprised of denaturation at 95 °C for 3 min followed by 50 cycles of amplification .Total RNA was prepared from 50 defective or normal embryos (Invitrogen), with amplification of 3 μl of 1st-strand cDNA. Amplification primers for each specific mRNA deduced from published sequences were tbx5-MO (5-GAAAGGTGTCTTCACTGTCCGCCAT-3), was designed against the tbx5 translational start site . Wild-type (WT) embryos, primarily at the 1-cell stage with the chorion intact, were injected with 19.4 ng/4.3 nl of stock MO diluted in Danieau’s solution. Injected embryos were raised at 28.5 °C. Embryos used for analyzing the expression of various markers were fixed with 4 % paraformaldehyde. Otherwise, embryos were scored after 2 days of development for late effects. In our previous study, 4 control groups, including the 3' end of tbx5-MO(2) (5’-GCCTGTACGATGTCTACCGTGAGGC-3’), mismatched tbx5-MO (5’-GTCTCTTGACTCTCCGCGATCTCGG-3’), and embryos with blank microinjection and wild-types without microinjection, were included to identify the specific blockage of tbx5 mRNA translation effect of tbx5-MO [tbx5-MO has been confirmed in previous published articles [The MO antisense oligonucleotide, articles .n = 150/group, with triplicate determinations), accompanied by 19.4 ng/2.3 nl tbx5-MO at the 1-cell stage. Treated zebrafish embryos were placed into plates with wells, and their functional classification was evaluated at 30, 48, 72, and 96 h post-fertilization (hpf).Zebrafish embryos were micro-injected with 1 fM of human GH  . Whole-mount in situ hybridization was carried out essentially as described by Oxtoby and Jowett [amhcvmhc, or cmlc2 probes at 67 °C. An alkaline phosphatase-conjugated anti-digoxigenin antibody  was used to detect zebrafish amhcvmhc, or cmlc2 signals. After staining with NBT/BCIP (Boehringer Mannheim), embryos were re-fixed with 4 % paraformaldehyde and stored in phosphate-buffered saline (PBS).Whole-mount r et al. . The digd Jowett . In brieZebrafish embryos were fixed with 4 % paraformaldehyde in PBS. De-paraffinized sections (3 μm) of zebrafish embryo tissues were placed on slides and processed for immunohistochemistry. After application of a biotin blocking system  for 30 min, sections were incubated with target-purified rabbit primary antibodies, including Bcl2, Bad, Cdk2, and P27  washed in PBS, and then incubated with a rhodamine-conjugated secondary antibody, goat anti-rabbit immunoglobulin G (IgG). After washing in PBS, sections were incubated with mounting medium and kept at 4 °C.4 in Sorenson’s phosphate buffer followed by dehydration through a graded series of ethanol washes, and embedded in Spurr’s EPON. Blocks were heated in an oven for 8 h at 70 °C. Semi-thin (1 μm) sections were cut and stained with toluidine blue for adequate preview under a microscope. Ultrathin sections (900 Å) were cut with a diamond knife, stained with uranyl acetate and lead citrate, and examined with an electron microscope.Embryos were fixed at 48 hpf with 2.5 % glutaraldehyde in Sorenson’s phosphate buffer, post-fixed with 1 % OsOBoth whole mount and sectioned TUNEL assays were performed using an ApopTag kit . Zebrafish embryos were briefly fixed with 4 % paraformaldehyde in PBS. Proteinase K-treated whole embryos or de-paraffinized sections (5 μm) of embryos were incubated with the TdT enzyme followed by anti-digoxigenin. Finally, embryos or slides were stained with DAB for 5 min.Embryos were homogenized on ice in lysis buffer (Sigma-Aldrich). Cellular debris was then pelletized by centrifugation at 12,000 rpm for 20 min, and the supernatant was collected and measured. Proteins were mixed with sample buffer before separation in 10 % sodium dodecylsulfate polyacrylamide electrophoresis (SDS-PAGE) gels. The SDS-PAGE was then transferred onto nitrocellulose membranes at 100 V for 1 h. Membranes were blocked with 5 % bovine serum albumin (BSA) buffer at room temperature for 1 h. The Akt and Erk primary antibody  was incubated overnight at 4 °C at a dilution of 1:1000. Nitrocellulose membranes were washed with PBST followed by incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000) for 1 h at room temperature before the images were digitized.p < 0.05.Results are given as the mean ± S.D. Where applicable, Duncan’s new multiple range test was used to compare every pair of testing groups. Statistical significance was accepted at tbx5-MO treated (MO) zebrafish embryos, along with curved trunks and abnormal “U-shaped” somites  groups. Formation of the heart  were observed in WT zebrafish embryos Figure A, their s Figure G, and pes Figure L was eits Figure P, trunk s Figure Q, and pes Figure R were vet Figure C, trunk t Figure H, and pet Figure M in the tbx5 using a zebrafish microarray. Genes that were 1.5× downregulated were included. Multiple genes participating in growth were downregulated in zebrafish embryos with the tbx5 deficiency  caused defects of the heart  caused specific defects of the heart   and either the MIS group (n = 50) or the WTGH group (n = 50).Compared to the WT group, microinjection of exogeneous GH into WT embryos (WTGH) did not cause significant phenotypic changes, and no embryonic defects were identified Figure P-R. Therigf1 RNA expression analysis in the heart and pectoral fins , and ghra[tbx5 in embryos diminished GH/IGF-1 pathway mediators, including GH, IGF-1, and GH receptors.Significant dysmorphogenesis Figure  shown inficiency -35. A de and ghra,36. Our via intercellular substance. The expression of igf1ghraghrberk1, and akt2 genes was increasing after co-injection with GH and tbx5 morpholino at 1-cell stage. These results revealed the exogenous GH has activated the downstream signaling pathway. However, it is still unknown how and where the exogenous GH binds with the GH receptors. Whether the injected exogenous human GH is packaged and sent outward to bind with the membranous GH receptors, or it directly binds with the cytoplasmic GH receptors, remains unidentified because the expression of GH receptors could be either in the nucleus or cytoplasm, or both, in different embryonic tissues and cells [Exogenous GH was used in our study to partially restore the anomalies during embryogenesis to ensure the involvement of GH in multiple organ defects by tbx5 knockdown. In the study, we injected human recombinant GH, which human GH instead of zebrafish GH is reported to have full activity in fish as well as in human , into thnd cells .tbx5 deficiency could activate expression of GH receptor genes, to induce an increase in igf1 levels, and to elevate downstream Akt and Erk systems, coinciding with restoration of morphological anomalies and transcriptional cascades. It could be hypothesized that GH is a factor that may act in a paracrine manner within the tbx5 functional pathway to modulate embryonic development.Exogenous GH in zebrafish embryos with Tbx5 is essential for regulating the progression of the cell cycle by controlling the length of the embryonic cardiac cell cycle [septum primum[tbx5 downregulated genes, which contributes to developmental delays in organogenesis including the cell cycle (p27 and cdk2) and apoptosis (bcl2 and bad). Early administration of exogenous GH improves the outcome of tbx5-deficiency-mediated heart defect embryos probably by inducing cardiac cells to re-enter the cell cycle. It also reduces aberrant apoptosis because GH works in a similar way to stimulate the cardiomyocyte to re-enter the cell cycle and thereby increases the number of cardiac myocytes in ischemic and infarcted myocardia [ll cycle  and reguum primum, all of um primum. In our yocardia ,26.cmlc2, amhc, and vmhc). Exogenous GH restored the expression levels of amhc, vmhc, and cmlc2 in our tbx5 morphant embryos with cardiac defects. Inactivation of tbx5 diminished amhc, vmhc, and cmlc2 expressions, and although it also reduced heart size, exogenous GH reversed that result and enhanced cardiac formation in zebrafish embryos. Our results indicate not only that GH is necessary for the growth of these cardiac structures, but also that supplementary exogenous GH restores tbx5 knockdown-mediated defects, including dysmorphogenesis and cardiomyogenesis, via cell proliferative and apoptotic pathways. It could be concluded that knockdown of tbx5 in early zebrafish embryogenesis causes functional GH deficiency and leads to dysmorphogenesis. The comorbidity of morphologic defects and functional GH deficiency could be observed in early embryogenesis of tbx5 morphants and implied that GH may involve a role in embryogenesis including cardiomyogenesis through transcriptional regulation of tbx5.The GH signaling pathway governs cell growth, proliferation, and apoptosis by controlling key regulatory genes that execute these processes. Herein, we also provide the first evidence that tbx5 acts together with GH to regulate cardiac myogenetic pathway-responsive genes  without subsequent effects, but the phenomenon didn’t happen in the expressions of ghra, ghrb, erk1, and akt2. It may aggressively assume that GH effects may involve IGF-1 dependent and independent pathways in embryonic development to switch on the cascade reactions. Thus the role of IGF-1 attracts attention for further exploration. Moreover, the expressions of ghra and ghrb in WTGH group significantly increased than MOGH group, but the expressions of erk1, akt2, amhc, vmhc, cmlc2 in WTGH group had no remarkable change than MOGH group. It implies that excess GH works inefficiently in individuals without tbx5 deficiency, or GH deficiency. GH receptors could be reactivated and responded to exogenous GH, but downstream signals and cardiomyogenesis-related genes didn’t markedly act in response to overload of GH. The results are compatible with the biological functions of GH in mature adult individuals. Surly, supplementary designs of control groups, for example, such as use of BSA with equivalent amount as a blank control, could be launched to access the authentic GH effects by our experimental model.We established four control groups in order to verify the specific GH effects without interference of morpholino and technical influences of micro-injection. However, interesting phenomena were disclosed by careful interpretation among those different control groups. First, tbx5 morphants, it cannot completely rescue those changes. Proper timing of GH treatment and optimal dosing might be found to enhance its therapeutic capability. Conversely, this approach may be limited by GH being partially significant to the complex functioning of the tbx5 network or because it compensates for only a small part of the chronological effects of tbx5 deficiency. Additional research is required to determine whether it is practicable to introduce GH to mend developmental defects in early embryogenesis.Although GH is able to partially restore dysmorphogenesis and cascade gene expressions in tbx5 deficiency. We suggest that the functional knockdown of zebrafish tbx5 results in a failure to develop a complete or functional heart, trunk, and pectoral fins and might be due to a functional GH deficiency induced by the tbx5 deficiency because it is a key factor causing abnormal organogenesis. Exogenous GH experiments in zebrafish embryos with tbx5 deficiency led to the conclusion that intrinsic growth-control mechanisms, including apoptosis, cell cycle, and cardiomyogenesis that control organic growth, depend on local GH and growth factors between cells and their neighbors. They also indicate that these interactions include controlling cardiac loop formation and the development of trunk and pectoral fins. The improvement of abnormal embryonic organogenesis in zebrafish embryos with tbx5 deficiency by the administration of exogenous GH suggests its potential application in human congenital anomalies.In summary, our work provides novel insights into the possible role of GH in contributing to developmental defects in zebrafish embryos with The authors declare that they have no competing interests.JHL and JKL conceived of the study, participated in its design, coordination. TCT participated in its design and drafted the manuscript. SLC and SYY carried out the molecular genetic studies. RBT and HYL participated in its design and coordination. All authors read and approved the final manuscript.


Beta diversity, which involves the assessment of differences between communities, is an important problem in ecological studies. Many statistical methods have been developed to quantify beta diversity, and among them, UniFrac and weighted-UniFrac (W-UniFrac) are widely used. The W-UniFrac is a weighted sum of branch lengths in a phylogenetic tree of the sequences from the communities. However, W-UniFrac does not consider the variation of the weights under random sampling resulting in less power detecting the differences between communities.We develop a new statistic termed variance adjusted weighted UniFrac (VAW-UniFrac) to compare two communities based on the phylogenetic relationships of the individuals. The VAW-UniFrac is used to test if the two communities are different. To test the power of VAW-UniFrac, we first ran a series of simulations which revealed that it always outperforms W-UniFrac, as well as UniFrac when the individuals are not uniformly distributed. Next, all three methods were applied to analyze three large 16S rRNA sequence collections, including human skin bacteria, mouse gut microbial communities, microbial communities from hypersaline soil and sediments, and a tropical forest census data. Both simulations and applications to real data show that VAW-UniFrac can satisfactorily measure differences between communities, considering not only the species composition but also abundance information.VAW-UniFrac can recover biological insights that cannot be revealed by other beta diversity measures, and it provides a novel alternative for comparing communities. The assessment of differences between communities is an important problem in ecological studies. By comparing the compositions of natural communities from different environments, locations or time periods, we can learn how specific factors affect community assembly and how species or individuals associate with each other -3. The dMany statistics have been proposed to compare communities based on genomic sequence data of a specific gene sampled from the communities. These include LIBSHUFF , ∫-LIBSHLozupone et al. proposed a novel statistical method termed UniFrac  and a weDespite the many studies on statistical methods to compare communities, there had been some confusions about the hypotheses being tested for the different statistics. Schloss  addresseNote that in W-UniFrac the length of a branch is weighted by the difference of relative abundances of the two communities for that branch of the tree. Under the null hypothesis that the two communities are the same, the weights for the different branches in W-UniFrac have difference variances and we provide a formula for calculating the variance in this paper. Based on the variance formula, we propose a new weighting scheme for the branch length in W-UniFrac by taking the variation of the weight into consideration. The new resulting statistic is termed Variance Adjusted Weighted UniFrac (VAW-UniFrac). The statistical significance of VAW-UniFrac is evaluated by randomizing the labels of the sequences along the leaves of the tree. Similar to UniFrac and W-UniFrac, the VAW-UniFrac can be used to evaluate if two communities are different. More precisely, it tests the hypothesis that the sequences from the communities are randomly distributed along the leaves of the tree. To study the power of this new statistic, we first carried out simulation studies similar to that in  to detecGiven a phylogenetic tree composed of all individuals from two communities, UniFrac distance  is definn is the number of branches in the tree, i bis the length of branch i, i Aand i Bare the numbers of individuals that descend from branch i in communities A and B, respectively, and T Aand T Bare the total numbers of individuals in communities A and B, respectively. In the denominator, n' is the number of different individuals in the two communities, j dis the distance from the root to individual j, while j αand j βare the numbers of times the sequences were observed in communities A and B, respectively . The same annotation will be used in the rest of the paper.In the numerator, Note thatWU is always larger than, or equal to, the numerator. Equality holds when either i A= 0 or i B= 0, which means that the two communities are totally separated. The numerators of both UniFrac and W-UniFrac can be written asTherefore, the denominator of In W-UniFrac,and in UniFrac,i in communities A, and Where i-th branch length assuming that the sequence labels are randomly distributed along the leaves of the tree. By ignoring the variance of i ωin W-UniFrac, the true relationships between communities may not be well characterized. Hence, we propose to adjust the weight i ωas follows. Given individuals from two communities, A and B, we first generate a phylogenetic tree composed of all the T A+ T Bindividuals in communities as leaves. Each leaf is labeled "A" or "B" to represent the community from which it comes. We test the hypothesis that the labels of the individuals are randomly distributed on the phylogenetic tree.From the definition of W-UniFrac given above, we note that it does not consider the variance of the weight i-th branch of the phylogenetic tree. Let i m= i A+ i Bbe the total number of individuals belonging to the i-th branch. Let m = T A+ T B. We randomly choose T Aindividuals from the total of m individuals, label them as being from community A and label the other leaves as being from community B. Then, iA, the number of individuals in community A that belong to the i-th branch, is hypergeometric with parameters  under the null hypothesis. That isConsider the Therefore,LetT Aindividuals are randomly sampled from the m individuals, we haveUnder the null hypothesis that the andi-th branch of the tree,From the above derivation, we propose the following variance adjusted weight (VAW) for the length of the We standardize the resulting statistic so that its value is between 0 and 1. The final VAW-UniFrac is defined asT , we also consider a variation by taking the weight as In addition to the statistic WU), VAW-UniFrac (T ) or SqT , are more powerful in detecting the relationship between two communities if they are related.Similar to UniFrac and W-UniFrac, the VAW-UniFrac aims to test if the two communities are different and, more specifically, if the sequences are randomly distributed along the leaves of the tree. The statistical significance of VAW-UniFrac is evaluated by randomizing the labels of the sequences. We are interested in which methods, including UniFrac, W-UniFrac  or the distribution patterns of samples in circles (ellipses) represented changing the differences between the communities. The maximum distance between any two points in one community was designed to be 0.3 units according to the distance between sequences from different phyla . Three cFor one comparison, we first sampled 200 points from each community, resulting in a total of 400 points. Second, Euclidean distances among all 400 points were calculated. Third, a phylogeny tree was generated based on this distance matrix using the neighbor joining method in the neighbor program in PHYLIP . The fourth dataset involves tropical forest census data in three plots across a precipitation gradient in central Panama [We applied UniFrac, W-UniFrac and VAW-UniFrac to reanalyze three datasets consisting of 16S rRNA sequences and one tropical forest census data. First, Costello et al.  investigr et al.  studied r et al. . All thel Panama -28. The l Panama . DiffereFor each dataset, we first calculated the distances between each pair of communities using the three statistics: UniFrac, W-UniFrac and VAW-UniFrac. Then the unweighted pair group method with arithmetic averages (UPGMA) clustering  was usedIn order to study our new methods and compare their performance to UniFrac and W-UniFrac, we carried out simulation studies according to the simulation methods developed in . We thenWe carried out three classes of simulations for two communities: 1) both were uniform samples from two circles with different centers and radii; 2) both were uniform samples from two ellipses with different orientations; and 3) one community was a uniform sample, while the other was an uneven sample from the same circle.In this simulation, communities were represented by circles, and individuals were uniformly sampled from each circle. It is reasonable to assume that the magnitude of a circle's radius can reflect the diversity of the community that it represents. In order to investigate the type 1 error rates and statistical power of the four statistics, UniFrac, W-UniFrac, VAW-UniFrac, and SqT, different levels of overlaps between communities were simulated by changing the distance between two centers, known as offset, and by changing the radius of one of the two circles  of bacterial 16S rRNA sequence data from Costello et al.  to underWe first assigned each sequence in samples to its closest relative in a phylogeny of the Greengenes core set  using BLFigure We wondered why the 8 native samples from individual F3 were clustered together and separated from other native samples of the same habitats according to VAW-UniFrac, while the clustering of native samples from F3 was not detected at all by UniFrac and W-UniFrac. To investigate this phenomenon in more depth, we then applied the three statistics to all 72 native microbiotas in that study. The UPGMA results are shown in Figure We then applied the three statistics to analyze the 16S rRNA sequences from mouse gut communities . LozuponAs observed in , both PCIn order to see how the statistics perform when they are applied to sequence collections derived by different technologies, but from the same sample, we analyzed the 16S rRNA sequence data from soil and sediments from . The samAfter applying the three statistics to each pair of the 16 samples, we used PCoA and UPGMA to analyze the results and investigated the performance of the three statistics  neighbor-joining, 2) maximum parsimony, 3) maximum likelihood, and 4) BLAST assignments of the sample sequences to the closet relatives on a known phylogenetic tree. The effects of the different tree construction methods on the results of UniFrac, W-UniFrac, and VAW-UniFrac are presented.Click here for file


Local curvature, or bending, of a graphene sheet is known to increase the chemical reactivity presenting an opportunity for templated chemical functionalisation. Using first-principles calculations based on density functional theory (DFT), we investigate the reaction barrier reduction for the adsorption of atomic hydrogen at linear bends in graphene. We find a significant barrier lowering (≈15%) for realistic radii of curvature (≈20 Å) and that adsorption along the linear bend leads to a stable linear kink. We compute the electronic transport properties of individual and multiple kink lines, and demonstrate how these act as efficient barriers for electron transport. In particular, two parallel kink lines form a graphene pseudo-nanoribbon structure with a semimetallic/semiconducting electronic structure closely related to the corresponding isolated ribbons; the ribbon band gap translates into a transport gap for electronic transport across the kink lines. We finally consider pseudo-ribbon-based heterostructures and propose that such structures present a novel approach for band gap engineering in nanostructured graphene. Nanostructures based on graphene have an enormous potential for applications. Especially in future electronic devices compatible with and extending silicon technology, due to the outstanding electronic transport properties of graphene . However3 hybridisation [A common approach towards engineering the electronic structure is to form quasi-1D graphene in the form of nanoribbons (GNR) . The eledisation . Periodidisation . Finallydisation , or a nadisation –10 can hdisation  down to disation .Graphene consists entirely of surface atoms and is thus exceedingly sensitive to the surroundings. In particular, the van der Waals (vdW) interaction with the substrate is of importance. The substrate interactions, which make graphene cling to small features, may be exploited by manufacturing nanostructures in the substrate. Periodic steps in a Cu substrate has been used to induce “wrinkles” or ripples in graphene with period and height on the order of 10 nm . Recentl2 nanoparticles induced local regions of strain and increased reactivity in a selective manner. Atomic hydrogen or other chemical species do not easily react with flat graphene when dosed from a single side [The ability to accurately control sharp local curvatures of graphene presents opportunities for strain-assisted modification of the local electronic structure and chemical reactivity in the graphene sheet, and may open a route to band gap engineered devices ,18–21. Vgle side . Howevergle side ,23. So fgle side –26. HoweIn this paper we consider the reactivity of linear bends in a graphene sheet, and the electronic transport properties of kinks resulting from the hydrogenation of bends. Our starting point is the generic graphene structure shown in L, while the rest of the atoms are allowed to relax. A separation of L = 90 Å is chosen in order to obtain realistic curvatures [The bend we consider in rvatures . We firsk-points. We use a mesh cut-off of 500 Ry throughout. When calculating reactivity in the form of reduced reaction barriers the unit-cell is chosen so that the distance between single hydrogen atoms is larger than 8.5 Å. This ensures low hydrogen–hydrogen interaction, which is known to impact reaction barriers [k-points). We use a TZP basis-set for hydrogen and a SZ basis for carbon, except in the reaction barrier calculations where we compare calculations using a DZP and SZP basis for the four carbon atoms nearest to the hydrogen. In the barrier determinations we furthermore use spin-polarised calculations because of unpaired electrons. For the relaxed geometries a force tolerance of 0.01 eV/Å is used, and the final energies are corrected for basis-set-superposition errors (BSSE) [The atomic and electronic structure calculations are based on density functional theory (DFT) using the SIESTA  code, anbarriers . In the s (BSSE) .S1 and S2 in S1 and S2. In the conductance calculations we employ a dense transverse k-point grid of 400 points.Based on the computed atomic and electronic structures we subsequently use the TranSIESTA  method tAdsorption of hydrogen on graphene involves a reaction barrier that needs to be overcome before the single hydrogen atom sticks to the graphene sheet. Several investigations based on DFT calculations show that atomic hydrogen adsorbs on-top on flat graphene with a barrier about 0.2 eV and binding energy in the range of 0.7–1.0 eV ,32–34. THere, we first focus on the trends in the change in adsorption barrier as a function of the local curvature of the graphene sheet. To this end we have considered atomic hydrogen absorption at the on-top carbon positions at points with different curvature on the bent structure, see 60 molecules, CNTs, and graphite to show that reactivity is increased with curvature. In our case we find that the local electronic density of states changes little for the atoms on the pristine bent graphene sheet (as in For the positions (I-VIII) we obtain the reaction barrier for adsorption of hydrogen as a function of the local radius of curvature (RoC) shown in et as in . Thus, w3-binding of a single H makes the graphene curve even more in its vicinity, which in turn, preferentially lowers the barrier for absorption of a H along the linear bend. This suggests a mechanism in which the hydrogen adsorption is propagating and leads to the decoration of the entire linear bend turning it into a kink line. It may be viewed as analogous to crack-formation mechanisms, where the breaking of a bond increases the stress on neighbouring bonds; only in this case, the graphene is hydrogenated rather than broken or destroyed.We may understand the reaction barrier and its change with curvature by considering the changes in carbon bond lengths. The barrier is due to the fact that the reacting carbon atom has to be pulled out of the graphene plane, stretching the strong carbon–carbon bonds, when reacting with the incoming hydrogen atom. When the graphene sheet is curved the carbon atom is already slightly out of the plane, and thus the energy required to pull the atom further out of plane is decreased compared to flat graphene. The ortho- and para-locations in the graphene hexagon have been shown to be the preferred locations for hydrogen adsorption in studies of flat graphene ,29,40. WS1 and S2 in 3 configuration where 2φ = 109.5°. The adsorption of H causes local changes in the geometry, i.e., only the carbon atoms very close to the kink are moved, while the remaining structure remains unperturbed. For this reason, the adsorption of hydrogen atoms can be considered as a process that locally pins the bend.Next, we examine the energetic and transport properties of kink-lines in the armchair direction. We first consider a single kink with angle φ, e.g., between sections E = 0), e.g., T0E. We find similarly that the calculated kink-transmission curves are also linear, and therefore we express the results for the transmissions in terms of the roughly energy-independent ratio T/T0 = const. The kink breaks the electron–hole symmetry, and we fit E > 0 and E < 0 separately, as shown in T/T0 is close to 0.17 in both regions . The close correspondence between the electronic band structure for the GNR and the transmission gap for the double-kink system allows us to consider the structure between two kinks as a pseudo-ribbon.Band-structure calculations show that periodic nanoscale rippling of the graphene is not sufficient to create a band gap  due to t integer . In FiguEgap = 0.4 eV and Egap = 0.5 eV, are in reasonable agreement with the power-law scaling of Egap with width found for aGNRs [N = 18 case a larger van Hove resonance is seen at the valence band edge, while for N = 19 a larger resonance is seen at the conduction band edge. There are small transmission values within the electronic band gap due to leakage through the barriers, which we expect to introduce shifts in the energies between the real and pseudo aGNR.For the semiconducting pseudo-ribbons transport gaps surrounded by van Hove-type 1D behaviour are seen in the transmission functions , in the band structure along the pseudo-ribbon direction. The weight on S3 is represented by a circle of radius Rnk,In order to analyse the transmission we single out the band structure projected on to the top section, kx (kz = 0) with n (i) being the band  index. The obtained projected band structures are shown in the left parts of each subfigure in S3 and other sections.Here Ψ is the wave function at S2/S3/S4 being M/M/M and SC/M/SC, respectively. For the all-metal pseudo-ribbons, M/M/M, an almost energy-independent transmission function is seen with a transmission close to that of the metallic double-kink in the previous subsection. The SC/M/SC structure shows a transport gap similar to that of the single SC pseudo-ribbon with van Hove resonances, while the S3-projected band structure reveals isolated metallic states within the gap. We note that the transmission at the resonances for the SC/M/SC structure is larger than the corresponding M/M/M transmission. For the case of semiconducting top pseudo-ribbons in S2, S4, i.e., there is a good correspondence between the side section band gaps  and the transmission gaps.In The presented investigations show that linear kink-line structures may form in graphene by reacting with atomic hydrogen along a linear bend in the sheet. The adsorption barrier is lowered in the close vicinity of the bend, which can be exploited to form the kink. In particular, we have shown that a radius of curvature of ≈20 Å reduces the hydrogen adsorption barrier by roughly 16% compared to H adsorption on pristine graphene. The calculations suggest that once a single hydrogen atom has been adsorbed, the induced local kink and resulting increase in local curvature makes it easier for the following H to adsorb, thus creating a propagating kink formation. A full line of hydrogen atoms pins the structure and divides the electronic systems into different regions. We have shown that the electronic transmission through a single kink is reduced by 83% compared to pristine graphene, meaning that the kink-line acts as an efficient barrier for electron motion. We have demonstrated how two close-by parallel kinks form a pseudo graphene nanoribbon with similar behaviour of the electronic structure to that for isolated nanoribbons. The transmission function displays transport gap features corresponding to the isolated nanoribbon band gaps.The present work thus suggests that it may be feasible to template functional electronic nanostructures by using the conformation of graphene, e.g., to the substrate, and that this in turn induce changes in local reactivity. Our work clearly calls for extensions in a number of directions. First of all more calculations are needed in order to investigate the kink-line propagation reaction proposed by our results. To this end it is important to include a realistic description of the actual substrate. It is also interesting to consider other adsorbate species, possibly introducing doping of the pseudo-ribbons and electronic gating. Finally, decoration and pinning of the edges of other geometries such as “bubbles” or “blisters” is of interest, e.g., in order to produce GAL-like structures  without