Ligusticum chuanxiong, has been reported to be effective in preventing cerebral ischemic stroke (CIS). In this study, we employed network pharmacology to reveal potential mechanism of SEH against CIS on a system level and confirmed the therapeutic effects of SEH on CIS by models of cerebral ischemia-reperfusion in vivo and in vitro. Through protein-protein interaction networks construction of SEH- and CIS-related targets, a total of 62 key targets were obtained by screening topological indices and analyzed for Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment. Gene Ontology analysis indicated that SEH might have a role in treating CIS via regulating some biological processes including regulation of transcription from RNA polymerase II promoter, epidermal growth factor receptor signaling pathway, phosphatidylinositol-mediated signaling, and some molecular function, such as transcription factor and protein phosphatase binding and nitric oxide synthase regulator activity. Meanwhile, the Kyoto Encyclopedia of Genes and Genomes analysis showed that phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway was significantly enriched. In addition, our result showed that SEH posttreatment significantly decreased the neurological scores, infarct volume, and neuronal death in the middle cerebral artery occlusion mice. Moreover, the PI3K/Akt/nuclear factor kappa B signaling pathway was activated by intragastric administration of 40 mg/kg SEH, as verified by Western blot. In vitro, treatment of PC12 cells with 100 μM SEH markedly reduced cell death induced by oxygen-glucose deprivation through the activation of PI3K/Akt/nuclear factor kappa B pathway, and the therapeutic effect of SEH was obviously inhibited by 10 μM LY294002. In summary, these results suggested that SEH carries a therapeutic potential in CIS involving multiple targets and pathways, and the most crucial mechanism might be through the activation of PI3K/Akt/nuclear factor kappa B (NF-κB) signaling pathway to inhibit inflammatory factor releases and increase the antiapoptosis capacity. Our study furnishes the future traditional Chinese medicine research with a network pharmacology framework.Senkyunolide-H (SEH), a major bioactive compound extracted from  Stroke is reaching epidemic proportions in the world, marked by high incidence, death, and disability rates . Responsin vitro and in vivo via activating the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) and mitogen-activated protein kinase (MAPK) pathways  and input onto the PharmMapper server (http://lilab.ecust.edu.cn/pharmmapper/) in a MDL sdf. format file, which was designed for identifying possible targets of small molecules via a reverse pharmacophore matching approach , which is the central hub for collecting protein function information with exact, consistent, and abundant annotation (The structure of SEH (PubChem CID: 10036567) was downloaded from the NCBI PubChem database (approach . The spenotation .http://www.omim.org) and DisGeNET database (http://www.disgenet.org/). We used “cerebral infarction” and “cerebral ischemic stroke” as the keywords, respectively, for genes searching, respectively  were applied for Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of key targets. This involves a two-step operation: first, the ID of gene was inputted and the species was specified as “Homo sapiens;” second, enrichment analysis was executed using functional annotation tool. Three GO enrichment categories of biological process, molecular function, and cellular component were selected to draw bar charts. A bubble chart of top 20 pathways was made by OmicShare platform (http://www.omicshare.com/).The Database for Annotation, Visualization and Integrated Discovery (DAVID)  supplied 8-week-old male Institute of Cancer Research mice (25-30 g), and all mice were domesticated for 7 days at least. The environment was maintained at room temperature of 23°C, humidity of 60%, and adequate free forage and water. To simulate a normal growing environment, we made a light-dark cycle that changes every 12 h. All experimental procedures were carried out in accordance with the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Yangzhou University-Institutional Animal Care and Use Committee (YIACUC-14-0015) based on ethical procedures and scientific nursing.Isoflurane (3-4%)  in 30% oxygen and 70% nitrous oxide was used to anesthetize the animal. Then, the right common carotid artery, vagus nerve, external carotid artery, and internal carotid artery were exposed and separated carefully after a median incision of the neck skin. A monofilament nylon filament was inserted into the internal carotid artery through an incision of external carotid artery until the middle cerebral artery (MCA) occluded and taken out to commence reperfusion after 45 min infraction. Sham-operated groups were given the same surgery without inserting the filament. The whole surgery would maintain the body temperature of mice at 37.0-37.5°C by a rectal thermometer connected to an automated heating pad . Thereafter, the mice were incubated in the thermal incubator to maintain the body temperature up to euthanasia.n = 14 in each group): sham operation (sham group); sham operation with 40 mg/kg SEH treatment (sham-SEH group); MCAO treatment (MCAO group); MCAO with 20 mg/kg SEH treatment (20 SEH group); and MCAO with 40 mg/kg SEH treatment (40 SEH group). SEH was administered intragastrically at the start and end of ischemic surgery immediately. Equal volume of 0.9% saline was intragastrically injected into two groups of mice (sham and MCAO). Mice were killed at 6 and 24 h after reperfusion. SEH  was dissolved in 0.9% saline and 0.1% dimethyl sulfoxide, and the purity was above 98.32%.According to previous studies , 25, 26,The neurological function of mice was assessed in accordance with the Bederson scale before killing. Four levels were divided in Bederson scale : level 0The procedure is following that described above . The micWe collected the tissues according to the method described in the previous study , and 10%2O2) diluted in PBS for 20 min and 5% normal serum diluted in 0.01 M PBS for 30 min. Then, the slices were incubated successively with diluted rabbit antineuronal nuclei (anti-NeuN)  overnight at 4°C, biotinylated goat antirabbit IgG , and streptavidin peroxidase complex . After that, the tissues were dyed with 3,3′-diaminobenzidine tetrahydrochloride in 0.01 M PBS and dehydrated on the adhesion microscope slides. Finally, we used neutral gum  sealing piece. Digital images of CA1 region were observed with a computer-based microscope , which is equipped with an image-analyzing system. Cell counts were gained through averaging the counts from the sections of each animal. The staining intensity of NeuN immunoreactive structures was assessed on the basis of an optical density (OD), which was obtained after the transformation of the mean gray level using the formula: OD = log (256/mean gray level). The OD of the background was taken from areas near the measured area. After the background density was subtracted, a ratio of the OD of image file was calibrated as percent [relative OD (ROD)] through Adobe Photoshop version 8.0 and then analyzed by NIH Image 1.59 software. We normalized each sample against the level of vehicle-sham sample. All measurements were performed under the same conditions by two observers in blind conditions to ensure objectivity.Immunohistochemistry was conducted according to our formerly published program . The sli6 cells/well in six-well plates and cultured at 37°C in a damp atmosphere containing 5% CO2. Cells were grown in Dulbecco's modified Eagle's medium (DMEM)  included penicillin (100 U/ml), 5% heat-inactivated fetal bovine serum (Gibco), streptomycin (100 mg/ml), and 10% horse serum (Gibco). To simulate the model of cerebral ischemia in vitro, cells were incubated in the serum/glucose-free DMEM after being seeded overnight and washed twice with PBS (pH 7.2) and then transferred to a hypoxic chamber  containing 95% N2 and 5% CO2 for 6 h. After oxygen-glucose deprivation (OGD) exposure, the cells were incubated with conditioned DMEM at 37°C in a damp atmosphere containing 5% CO2 for 24 h reperfusion. In addition, the cells were divided into different groups: the sham group, the OGD group, the OGD + SEH groups , and the OGD + SEH + LY294002 (10 μM) groups. The different concentrations of SEH and LY294002  were added throughout the OGD-reperfusion (OGD/R) treatment within the standard medium. The vehicle group was subjected to the same experimental procedures without exposure to the serum/glucose-free DMEM medium and any drugs. Then, the cells were collected for Western blotting detection.PC12 cell line (rat pheochromocytoma) was purchased from the American Type Culture Collection, seeded at 1 × 103 cells/100 μl. After stabilizing, cells were exposed to OGD/R in the absence or presence of indicated concentrations of SEH and LY294002 treatment. After 24-h reperfusion, 10 μl of CCK8 solution was included to each hole (100 μl medium), incubated at 37°C for 2 h, and then, the absorbance was measured at 450 nm in a multimode plate reader .Cell Counting Kit-8 was used for measuring cell viability . Cells were seeded into a 96-well plate at a density of 6 × 10n = 7 in each group) were cut into 400-μm thickness serially and transversely on a vibratome, and the hippocampus was carved with a scalpel. We preprocessed the tissues and the whole cell lysates from PC12 cell by Whole Cell Lysis Assay kit /Total Protein Extraction Kit and measured the protein concentration with a Pierce BCA Protein Assay Kit . The same amount of protein (40 μg) was separated by suitable percentage sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes . For a clear band, the membranes were cultured in Tris-buffered saline containing 5% bovine serum albumin and 0.1% Tween 20 for 60 min, then incubated in primary antibody overnight at 4°C, and followed in the corresponding secondary antibody at room temperature for 2 h. Protein expression was detected by SuperSignal West Pico chemiluminescent substrate .Western blot experiment was carried out on the basis of a procedure published before . The micDensitometric analysis on all scanned Western blot results was performed using Quantity One Analysis Software (Bio-Rad). Relative OD (ROD) calculations are mainly followed: the ratio of the calibrated ROD is expressed in percentage, and the sham group is defined as 100%. Three similar independent experiments were represented by each blot results at least. The primary and secondary antibodies were listed as follows: rabbit anti-PI3K  and p-PI3K , rabbit anti-Akt  and p-akt , rabbit anti-NF-κB , rabbit anti-Bcl-xL , rabbit anticleaved caspase 3 , anti-β-actin , and goat antirabbit IgG .p < 0.05 was considered to have statistical significance.The data shown in the study represent the means ± SD and were plotted in histograms with GraphPad Prism 7.0. Differences in average values among groups were analyzed through one-way analysis of variance test by SPSS software. A total of 11 potential targets were derived and screened with a fit score value >4.5 from the top 100 potential human protein targets of SEH through PharmMapper server. Detailed information is described in As the charts show , 4, SEH GO analysis of 62 candidate targets for SEH treating on CIS was performed via the DAVID database to investigate their functions and inherent significance in the biological system networks. The diagram  containep < 0.01 by means of DAVID database  signaling pathways. Gene transcription regulation is a vital process in CIS. It is coordinated by transcription factors and other proteins and regulates gene functions via a variety of mechanisms, for instance, neurogenesis, inflammation, and angiogenesis . Recent In KEGG analysis, PI3K/Akt, ErbB, neurotrophin, and FOXO signaling pathways were significantly enriched, and PI3K/Akt signaling pathway may be the most crucial mechanism. The PI3K/Akt pathway is the key to ErbB signal transduction, which controls protein homeostasis through boosting migration, angiogenesis, and cell proliferation. Zhou's data showed that reactive oxygen species eliminated the inhibitory effect of PI3K/Akt signaling pathway on ERK activity during reperfusion, and the strong activation of ERK activity played a pivotal role in cell injury induced by reperfusion . In addiin vivo and in vitro through the activation of Nrf2 and CREB via PI3K/Akt signaling, which could be reversed by cotreatment with LY294002 .JZ and YJ performed the experiments and drafted the manuscript. YJ, NL, TS, HJ, and JL analyzed the data. BY designed the experiments. All the authors discussed the results, reviewed the final manuscript, and approved it for the publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We propose a molecular generative model based on the conditional variational autoencoder for de novo molecular design. It is specialized to control multiple molecular properties simultaneously by imposing them on a latent space. As a proof of concept, we demonstrate that it can be used to generate drug-like molecules with five target properties. We were also able to adjust a single property without changing the others and to manipulate it beyond the range of the dataset.The online version of this article (10.1186/s13321-018-0286-7) contains supplementary material, which is available to authorized users. The ultimate goal of molecular design for new materials and drugs is to directly generate molecules with the desired properties. This is apparently challenging work because a molecular space is extraordinarily vast, discrete, and disorganized with diverse types of molecules. For instance, mentclass2pt{minimComputer-aided molecular design has attracted much attention as a promising solution to overcome the experimental limitation –6. Fast Recently emerging generative models based on deep learning techniques may offer a viable solution for more efficient molecular design. Gómez-Bombarelli et al. adopted a variational autoencoder  to optimWe note that various molecular properties are correlated with each other. Therefore, adjusting one target property by structural modifications may cause an undesired change in other properties. To avoid this problem in rational molecular design, one has to control several properties at the same time. Here, we propose a molecular generative model using the conditional variational autoencoder (CVAE)  suitableAs a proof of concept, we used the CVAE to generate drug-like molecules satisfying five target properties at the same time: molecular weight (MW), partition coefficient (LogP), number of hydrogen bond donor (HBD), number of hydrogen acceptor (HBA), and topological polar surface area (TPSA). We were able to produce a number of molecules with the specific values of the five target properties within a given range. It was also possible to adjust a single target property without changing the others. Furthermore, we were able to generate molecules with properties beyond the range of the database.E denotes an expectation value, P and Q are probability distributions, X and z indicate the data and latent spaces, respectively. The first and second terms are often called the reconstruction error and the KL term, respectively. In an autoencoder, Q(z|X) and P(X|z) are approximated by an encoder and a decoder, respectively. A key difference of the CVAE from the VAE is to embed the conditional information in the objective function of the VAE, leading to the revised objective function as follow:c denotes a condition vector. The condition vector c is directly involved in the encoding and decoding processes. In our model, the molecular properties we want to control were represented as the condition vector. As a result, the CVAE can generate molecules with the target properties imposed by the condition vector.We selected the CVAE as a molecular generator. It is one of the most popular generative models which generates objects similar to but not identical to a given dataset. In particular, it is distinguished from the VAE in that it can impose certain conditions in the encoding and decoding processes. To elucidate the difference between VAE and CVAE, we compared their objective functions with one another. The objective function of the VAE is given byIncorporating molecular properties in the VAE to generate molecules with desirable properties are also possible through a two-step model proposed by Gómez-Bombarelli et al. In this method, the VAE is trained jointly with an additional neural network for property prediction. Subsequently, a Gaussian process model creates a mapping from the resulting latent space to the associated molecular properties. Finally, property optimization in the resulting latent space is performed by a gradient descent optimization method.The key difference of our CVAE model from the jointly trained VAE model is that the molecular properties are directly incorporated into both the encoder and decoder. The resulting latent vector is composed of two parts: the first part is for the target molecular properties, while the second part involves the molecular structures and the other properties. Therefore, the desired molecular properties can be embedded in a target molecular structure simply by setting a condition vector. In other words, one can control the structure and the properties independently except for some cases in which the properties are strongly coupled to a molecular scaffold. This is particularly useful to incorporate a certain property in a given molecule just with a marginal structure modification. After all, the CVAE is less sensitive to the continuity and smoothness of the latent space, because it does not require the derivative of the latent space with respect to the latent vector of the molecular structure. Another technical difference of the CVAE from the jointly trained VAE is that it does not need any further optimization process, which is inevitable in the jointly trained VAE for each different property value.We represented molecules with SMILES codes to take advantage of state-of-the-art deep learning techniques that are specialized in dealing with texts and sequences. Each SMILES code was canonicalized for a unique molecular representation. One ‘E’ was padded on the end of the SMILES code to indicate the end of the string. Subsequently, each character including ‘E’ is represented with a one-hot vector, resulting in an input matrix. Each one-hot vector of the input matrix is transformed to an embedding vector with the size of 300, and then the input matrix is concatenated with a predefined condition vector. The first, second, and last entries of the condition vector are filled with information consisting of the MW, LogP, and TPSA, respectively, while the remaining two entries are labeled by the HBD and HBA as shown in Fig. The resulting matrix is subjected to the encoder of the CVAE to generate a latent vector. We adopted the so-called recurrent neural network (RNN) with an LSTM cell for both the encoder and decoder of the CVAE . They arRDKit , an openhttps://github.com/jaechanglim/CVAE).The total dataset is made of molecules randomly selected from the ZINC dataset . GeneralAs the first application, we demonstrated that the CVAE method can generate molecules with specific values for the five target properties by applying it to Aspirin and Tamiflu. The values of the  for Aspirin and Tamiflu are  and , respectively. The condition vector of each molecule was made by those values. Latent vectors to be concatenated with the condition vector were sampled by adding a Gaussian type noise to the latent vector of a molecule selected randomly in the training set. Figure The second application was to generate molecules similar in both properties and structure to the mother molecule by sampling latent vectors around that of the mother. Figure As the third case study, we tested whether the CVAE method can change only a single property without changing the others. The condition vector was constructed with the MW, HBD, HBA, and TPSA of Tamiflu, and we varied LogP from 0.0 to 3.0. Latent vectors were sampled around that of Tamiflu. Figure Finally, we investigated the possibility to change a specific molecular property beyond the range of a training set. Latent vectors were sampled around molecules in the training set. In the condition vector, the four properties were given randomly except for a single target property. The target property was set to 10% larger than its maximum value in the training set . Figure We analyzed the latent space constructed by the CVAE. Two principle axes were extracted by principal component analysis. Figure Apart from the successful applications of the CVAE method, it has a drawback that should be resolved. The success rate of generating desirable molecules is very low. We tested how many attempts were required to generate 100 molecules with the five desired properties and how many valid molecules were generated during those attempts. We also compared when the condition vector is set randomly or to target properties to show the effect of the condition vector for generating desirable molecules.Table We further analyzed the performance of the CVAE by investigating the change in the success rate and the number of valid molecules according to latent vector sampling methods. We employed three different sampling methods: random, around the latent vectors of known molecules, and around the latent vectors of target molecules. For all the sampling methods, the condition vector was constructed using the five properties of the target molecules. The generation process was continued until 100 molecules with the five target properties were successfully created from a single target molecule, and it was repeated for 100 different target molecules selected randomly from the ZINC dataset. Table We suspect that at some part the overall low success rates regardless of the latent vector sampling methods are due to the strong correlation between the five target properties. In addition, it is known that the discrete nature of SMILES causes a high rate of invalid molecules in the decoding process from latent vectors to molecules . The stoWe proposed a new molecular design strategy based on the conditional variational autoencoder. Instead of high-throughput virtual screening, our method as one of the deep learning-based generative models directly produces molecules with desirable target properties. In particular, its strength is controlling multiple target properties simultaneously by imposing them on a condition vector. We demonstrated that it was possible to generate drug-like molecules with specific values for the five target properties  within an error range of 10%. In addition, we were able to selectively control LogP without changing the other properties and to increase a specific property beyond the range of the training set. Thus, this new method has attractive applicability for efficient molecular design.Supplementary material 1 (docx 791 KB)Below is the link to the electronic supplementary material.


Avian influenza H5N1 has a high human case fatality rate, but is not yet well-adapted to human hosts. Amino acid substitutions currently circulating in avian populations may enhance viral fitness in, and thus viral adaptation to, human hosts. Substitutions which could increase the risk of a human pandemic  are termed key substitutions (KS). Egypt represents the epicenter of human H5N1 infections, with more confirmed cases than any other country. To date, however, there have not been any spatial analyses of KS in Egypt.Using 925 viral samples of H5N1 from Egypt, we aligned protein sequences and scanned for KS. We geocoded isolates using dasymetric mapping, then carried out geospatial hot spot analyses to identify spatial clusters of high KS detection rates. KS prevalence and spatial clusters were evaluated for all detected KS, as well as when stratified by phenotypic consequence.A total of 39 distinct KS were detected in the wild, including 17 not previously reported in Egypt. KS were detected in 874 samples (94.5%). Detection rates varied by viral protein with most KS observed in the surface hemagglutinin (HA) and neuraminidase (NA) proteins, as well as the interior non-structural 1 (NS1) protein. The most frequently detected KS were associated with increased viral binding to mammalian cells and virulence. Samples with high overall detection rates of KS exhibited statistically significant spatial clustering in two governorates in the northwestern Nile delta, Alexandria and Beheira.KS provide a possible mechanism by which avian influenza H5N1 could evolve into a pandemic candidate. With numerous KS circulating in Egypt, and non-random spatial clustering of KS detection rates, these findings suggest the need for increased surveillance in these areas. Highly pathogenic avian influenza H5N1 has a case fatality rate approximately ten times higher than the H1N1 “Spanish Flu” pandemic of 1918, but has much lower infectivity . Given tEgypt has reported more confirmed human cases of avian H5N1 than any other country, and is regarded by many observers as the epicenter of human H5N1 infections –17. HumaGenetic changes in viral proteins can have phenotypic consequences that increase pandemic risk . A numbehttps://www.ncbi.nlm.nih.gov/genbank/), we performed a descriptive spatial analysis to examine the distribution of KS in Egypt [Several KS have already been detected in Egypt, from samples at least as early as 2010 , 34, 35.in Egypt . A spatiin Egypt , 38. Impin Egypt . Areas wwww.cdc.gov/flu/avianflu/h5n1/inventory.htm). We used this inventory in the current study as the definitive list of known KS. KS have been identified in all 10 viral proteins, including the surface proteins HA and neuraminidase (NA) responsible primarily for virus binding, cell fusion, and viral release, and the internal proteins  involved in viral structure, replication, suppressing host antiviral response, and more [We obtained Egyptian H5N1 sequences collected between 2005 and 2015 from the Influenza Virus Sequence Database, hosted by the National Center for Biotechnology Information (NCBI) . We extrn = 29) are SNPs, such as the substitution from glutamic acid to lysine at position 627 (E627K) in the PB2 gene, shown to enhance mammalian host adaptation; two KS are amino acid deletions; and the remaining eight KS are sets of mutations , such as the dual substitutions of N200S in the NS1 gene and T47A in the NS2 gene, which together decrease host antiviral response but do not constitute KS individually. Table Most of the selected KS  were kept. We summarized results for each governorate by counting the total number of iterations in which at least one hot spot was detected. We considered governorates with 9500 or more detections to be probable hot spots. For comparison, we also performed a single hot spot analysis using each governorate’s geometric centroid, consistent with prior phylogeographic studies of influenza in Egypt [The majority of H5N1 samples from Egypt did not include geographic information below the governorate level to allow hot spot detection, consequently dasymetric adjustment was employed to approximate sample locations within governorates . We randilometer . This rein Egypt , 58. Fign = 50), Beheira (n = 96), Monufia (n = 90), Qalyubia (n = 109), Dakahlia (n = 82), and Sharqia (n = 88) , with an average of 3 PCGs represented. The majority of KS-positive samples (584/925) were located in Lower Egypt, particularly in the governorates of Alexandria (n = 825), followed by NA (n = 381); other viral proteins were much less frequent in the GenBank data. Detection rates varied by protein with the NA and NS1 proteins having one or more KS present in 100% of samples tested, while the PB1-F2 protein had only a single detected KS out of 88 samples tested. The average number of KS tested for was 22.6 per sample, with an average of 6.4 KS detected, giving an overall mean detection rate of 0.28. A total of 39 KS were detected at least once in H5N1 samples from Egypt. Table The most commonly sequenced protein was HA n = 85, followAlthough H5N1 was first reported in Egypt in 2006, 2 positive viral samples were collected in December 2005. The number of isolates uploaded to GenBank with governorate location information rose above 100 each year in 2007–2010, but dropped after 2011. Similarly, the average number of proteins sequenced per virus ranged from a high of 2.7 in 2008 to a low of 1.3 in 2014. Between 2005 and 2015, mean detection rates by year  ranged from a high of 0.38 in 2007 to a low of 0.18 in 2014. Figure n = 17), representing all 5 PCGs and including 5 of the 8 detected KS associated with antiviral susceptibility. One KS not previously reported in Egypt was a set of 7 amino acid substitutions in the PB2 gene, associated with both PCG 2 and PCG 3 [The single most commonly detected KS was the change in the HA protein at amino acid site 94 from aspartic acid to asparagine, associated with increased virus binding to human sialic acid receptors . KS werend PCG 3 . For a fTwo governorates contained probable hot spots of overall KS detection rates using both governorate centroids (single run) and dasymetrically-adjusted governorate boundaries (> 9500 iterations): Alexandria and Beheira. These two governorates are immediate neighbors in the northwestern portion of the Nile River Delta see Fig. . No othep-value cutoff of 0.05 to evaluate statistical significance. We detected probable hot spots based upon dasymetric adjustment for PCG1 in Alexandria and Beheira and for PCG2 only in Beheira, however the remaining PCGs falling below the 9500 iterations threshold. Results were not consistent between centroid and dasymetrically-adjusted analyses. Using a single iteration with all samples placed at governorate centroids, hot spots were found for PCG1 in Beni Suef, Dakahlia, and Faiyum; for PCG2 in Alexandria and Beheira; and for PCG4 in Asyut, Faiyum, and Monufia; while PCG3 and PCG5 again did not contain any hot spots  contained one or more KS, with 39 distinct KS detected including from each of the five PCGs. The circulation of so many KS increases the probability of recombination and reassortment producing viral genomes with combinations of KS that prove to be well-adapted to infect, replicate in, and transmit between humans.Samples with governorate location information uploaded to GenBank varied from year to year, with a low of 39 in 2005–6 and a high in 2009 with 155 isolates, however the sample sizes dropped after 2011. It is not entirely clear why fewer isolates from Egypt are being uploaded to GenBank than previously, although political turmoil and social unrest in the wake of the 2011 revolution are likely responsible. Detection rates of KS also varied, trending down during the study period from a high of 0.38 in 2007 to a low of 0.18 in 2014  provides an indication of the reliability of our estimates and does not assume all samples were collected at the geometric centroid of the governorate. Using dasymetrically-adjusted locations, spatial clusters of KS detection rates were identified only in Alexandria and Beheira for KS related to host specificity and virulence (PCG1 and 2).The city of Alexandria is the second largest city in Egypt and an important seaport, as well as a popular tourist destination. The neighboring governorate of Beheira is located between Alexandria and Cairo, experiencing substantial transport between those two urban centers. A study in Beheira in 2015 claimed the majority of the population was involved in raising poultry, and found most poultry growers demonstrated poor practices with regards to avian influenza prevention and control . SpatialA limitation of this analysis was that in most governorates sample sizes were too small to allow for time series analyses of detected hot spots, therefore spatial clusters were assumed to be consistent over the 10-year study period. We were also limited by the lack of fine-grain geographic location information for influenza sequences on GenBank, which were the primary source of data for this study. Recognized by previous researchers as an important shortcoming to the otherwise incredibly useful datasets available through GenBank, the lack of consistency and infrequent inclusion of sub-governorate location information required the use of approximation methods which introduced uncertainty into the analyses –72. PrevThis work provides a descriptive baseline for the distribution and spatial clustering of KS in Egypt, from which future studies can be compared. High overall detection rates of KS were found to cluster spatially in the northwestern portion of the Nile River delta. Large numbers of KS were detected with a range of associated phenotypic consequences, which raise concerns of a pandemic strain developing in the region. Better geographic data and improved genetic surveillance are necessary to properly evaluate and monitor this potential threat to global public health.


N-terminal was probed with anti-FLAG. A malachite green phosphatase assay to measure inorganic phosphate release from phospho-glucans, that is glycogen or LBs. Glycogen is used in this laforin bioassay as the biologically relevant substrate in order to determine the specific activity of the readthrough products. All reactions are incubated for 40 minute the absorbance is measured at 620 nm and the pmoles of phosphate released/min/nmol protein was calculated using a standard curve. RESULTS/ANTICIPATED RESULTS: HEK293 cells were transfected with MeCP2 R241X, laforin R241X, or laforin WT NT-FLAG construct, treated with different concentrations of gentamicin for 48 hours, and laforin levels were assessed by Western analysis with anti-FLAG. HEK293 cells were transfected with WT laforin or a laforin PTC CT-FLAG construct, treated with different concentrations of gentamicin for 48 hours, and laforin levels were assessed by Western analysis with anti-FLAG. B. Quantification of read-through for PTC experiments. *p-value≤0.001. #p-value≤0.001. Schematic of laforin bioassay. The assay has been performed with human and mouse tissue as well as cultured cells. B. Laforin bioassay results using laforin from PTC experiment. **p-value≤0.001. *p-value≤0.01. DISCUSSION/SIGNIFICANCE OF IMPACT: Our results suggest that gentamicin is not only responsible for inducing readthrough of the PTC mutations, but also for promoting translation of fully functional laforin. Therefore, our in vitro system for the analysis of PTC readthrough of laforin will be useful for determining which PTC mutations are suppressible with gentamicin or other small molecules, in what quantities laforin is recovered from PTC mutations, and if the protein products possess the appropriate enzymatic function.OBJECTIVES/SPECIFIC AIMS: A small molecule therapy is within reach to treat a molecular mechanism known to result in thousands of fatal diseases. For 10% of patients with a genetic disease, a nonsense/STOP mutation/premature termination codon (PTC) is the underlying cause of their malady. PTCs prematurely stop protein synthesis and yield truncated proteins. Truncated proteins typically provide little to no proper function or activity and are rapidly degraded; thus, disease is imminent. Recent work has demonstrated that small molecules including aminoglycosides can cause the ribosome to readthrough these PTCs. Thus, PTC readthrough with small molecules is a very attractive approach for treating diseases caused by PTCs. Small molecules that promote readthrough act on the ribosome and induce a ribosomal conformational change. In this conformation, the PTC is not recognized by the translational machinery and an amino acid is incorporated into the growing peptide chain, thus protein synthesis continues and does not stop. The use of a single small molecule to readthrough various PTC mutations has been repeatedly effective for in vitro studies and some of these have progressed to clinical trials. Although there has been success in defining these small molecules, the field has discovered that every PTC is unique and likely requires a different small molecule. Thus, developing a cell culture model to test read-through of Lafora PTCs and the functionality of the protein product is the first step to developing a readthrough therapy for a LD. METHODS/STUDY POPULATION: Method for in vitro quantification of readthrough: 24 hours before transfection, HEK293 cells were split in 6-well plates. On the following day, approximately 60% confluence, the cells were transiently transfected with the WT or PTC mutated constructs using Polyethylenimine HCl MAX. Cells were transfected with a total amount of 0.35 μg DNA/well and 2 μl Polyethylenimine HCl MAX/well. Four hours later, the transfection medium was removed and replaced with fresh medium, without streptomycin and penicillin. The fresh media contained gentamicin diluted to the indicated concentration per well. Fresh gentamicin-containing medium was replaced after 24 hours. After 48 hours, lysates were collected in 100 μL mRIPA supplemented with protease inhibitors for each construct. The lysates were run on a western blot and the


Background and Objectives: A coaxial electrospinning technique was used to produce core/shell nanofibers of a polylactic acid (PLA) as a shell and a polyvinyl alcohol (PVA) containing metformin hydrochloride (MH) as a core. Materials and Methods: Fish sarcoplasmic protein (FSP) was extracted from fresh bonito and incorporated into nanofiber at various concentrations to investigate the influence on properties of the coaxial nanofibers. The morphology, chemical structure and thermal properties of the nanofibers were studied. Results: The results show that uniform and bead-free structured nanofibers with diameters ranging from 621 nm to 681 nm were obtained. A differential scanning calorimetry (DSC) analysis shows that FSP had a reducing effect on the crystallinity of the nanofibers. Furthermore, the drug release profile of electrospun fibers was analyzed using the spectrophotometric method. Conclusions: The nanofibers showed prolonged and sustained release and the first order kinetic seems to be more suitable to describe the release. MTT assay suggests that the produced drug and protein loaded coaxial nanofibers are non-toxic and enhance cell attachment. Thus, these results demonstrate that the produced nanofibers had the potential to be used for diabetic wound healing applications. Wound healing is a complex, dynamic, continuous, and multicellular process which targets barrier repair in the skin. This process involves many factors, such as extracellular matrix (ECM), blood cells, growth factors, and cytokines. Thus, it is difficult to obtain excellent results in regeneration of the tissue due to the these factors and pathological and physiological complexity of this process ,2.Nanofibers can provide a favorable environment for wound healing due to their similarity to the extracellular matrix diameters, high porosity, high gas permeability, and high surface area-volume ratio. These advantages support skin regeneration, cell respiration, moisture retention, and hemostasis in the wound healing process ,4. The eThe coaxial technique is the electrospinning of simultaneous and coaxial capillaries to form the core-shell structure of two different components in nanofiber production . The nanNatural polymers  are widely used in nanomaterial production ,12,13. PThe use of synthetic polymers in combination with natural polymers results in higher mechanical properties and more flexible nanofibers . PolylacThe wounds caused by diabete disease are chronic, difficult to treat, and are most common in the feet . MetformAlthough many protein types, such as soy protein, whey, and keratin, have been used in drug release studies, there have been few empirical investigations into FSP protein in the use of drug delivery ,23. Therg, 5 °C. After the centrifugation, the supernatant was freeze dried  for obtain the FSP powder. This study was carried out under protocol number FEN-B-121218-0614 obtained on 15 June 2018.Fish sarcoplasmic protein (FSP) was extracted from fresh bonito by using the method presented in our previous study . The bonw/v) in DCM:DMF (4:1 v/v) and stirred for about 1 h to obtain viscous solution. PVA solutions at 5% (w/v) of polymer content were prepared in distilled water and stirred at 80 °C for 4 h to ensure complete dissolution. Then, the solution was cooled down to room temperature, 1 wt%, 3 wt% and 5 wt% FSP contents and 2% (w/v) metformin hyrochloride (MH) (20 mg) were individually added into the PVA solution under constant stirring. Contents of the core and the shell spinning solutions of the samples is given in PLA solution was prepared at a concentration of 8% . Two individual syringes containing the core (PVA solution) and the shell (PCL solution) were connected to a coaxial spinneret and driven by two separate pumps. A voltage of 13 kV was supplied by a high-voltage power supplier during coaxial electrospinning. The distance between the coaxial nozzle and the grounded collector was set as 12 cm, the flow rate of PLA solution was 1.4 mL/h, and the PVA solution was 0.5 mL/h. All of the electrospinning processes were carried out under ambient conditions (25 °C with a relative humidity of 55%).w/v) stacking gel and 12% (w/v) resolving gel according to the method of Laemmli (1970). An amount of 27.5 mg FD FSP sample was dissolved in 0.5 mL of demineralised water. It was then centrifuged and after centrifugation, the supernatant was used to determine the protein composition. The electrophoresis procedure was performed at 100 V for 15 min and at 120 V for 75 min by adjusting to pH 8.3 in tris glycine buffer. Samples were boiled for the denaturation procedure before the loading onto the gel. After the electrophoresis, proteins in the separating gel were determined by coomassie staining. Fermentas Protein Molecular Weight Marker  containing seven proteins within 14.4–116 kDa range was used in order to determine the molecular weight range of the proteins by the Image Analyzer System .The protein composition of the freeze-dried fish sarcoplasmic protein (FD FSP) was analyzed by a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) method using 5% (The morphologies of the electrospun fibers were examined using a scanning electron microscope(SEM)  at an accelerating voltage of 10 kV. Samples were coated with gold/palladium (18 nm) using a sputter coater . The average fiber diameters and their distribution were determined by the image analysis software  using the measurement of 100 fibers selected randomly from each sample.−1 to 4000 cm−1. The nanofibers used in the drug release study for 21 days were dried at vacuum oven at 40 °C for one day before the FTIR test.To determine the composition and possible interaction between the fibers components, the infrared spectrums of the fibers were registered using a fourier-transform infrared spectroscopy (FT-IR)  equipped with a Gladi attenuated total reflection (ATR) viewing plate  and a liquid-nitrogen cooled mercury cadmium telluride (MCT) detector at a wavelength of 500 cm2 and they were trypsinized and counted with a hemocytometer. All the nanofibers were cut to a size of 1 cm2 and sterilized in UV. DMEM cell culture medium containing 10% FBS, 1% Penicillin/Streptomycin as well as 100,000 cells were then seeded onto nanofibrous membranes in 24-well culture plate.The hacat cell lines, which is human keratinocyte cell line purchased from the American Type Culture Collection  were plated separately in tissue culture flasks and cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS). The cells were maintained in a humidified incubator at 37 °C with 5% CO2. After incubation, purple colored formazan crystal was formed at the bottom of the well and the above DMEM was withdrawn. DMSO solution was added to wells and measured for light absorbance in 96 wells/plate at 570 nm.MTT -2,5-diphenyl tetrazolium bromide) was dissolved in PBS at 5 mg/ml. The stock MTT solution (66 µl per well) was added on the cells and materials in DMEM supplemented with 10% FBS, 1% Penicillin/Streptomycin in the culture plate incubated for 2 h at 37 °C and 5% COIn vitro drug release was performed by the metformin hydrochloride which was loaded into the PVA/PLA electrospun nanofibers. The drug loaded nanofibers were cut into 2 × 2 cm pieces, weighted and immersed in glass bottles containing 7 mL of phosphate buffer solution (PBS) and the pH of the PBS solution was adjusted to 7.4 then shaked at 37 °C for 21 days. At determined time intervals, the amount of drug relase was measured at 233 nm by UV-VIS spectrophotometer. Each experiment was repeated three times.To disclose the drug release mechanism, the MH release profiles were fitted to four popular mathematical equations such as zero order, first order, Higuchi and Peppas-Korsemeyer equations represented as Equations (1)–(4).Q is the fractional amount of drug release at time t and “0K” is the zero-order release constant.Zero order kinetic defines a linear relationship between the fractions of drug release versus time:Q is the fractional amount of drug release at time t and 1K is the first-order release constant.The equation that describes first order kinetic isQ is the fractional amount of drug release at time t and hK is the Higuchi kinetic constant.Higuchi kinetic defines a linear dependence of the active fraction released per unit of square root of time:Q is the fractional amount of drug release at time t, K is the kinetic constant and n is the diffusion exponent which is indicative of the drug release mechanism.The equation of Korsmeyer–Peppas isThe thermal properties of the nanofibers were analyzed using a Perkin Elmer Jade differential scanning calorimeter (DSC). Samples were weighed and placed in alumina pans. The heating was performed from room temperature to 300 °C with a heating rate of 10 °C/min under dynamic argon atmosphere (20 mL/min).SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis technique) is commonly used technique for characterization of proteins. Molecular weight distrubition of FSP were determined. As illustrated in SEM images and associated diameter histograms of the prepared nanofibers having different concentrations of FSP are shown in 3 asymmetric stretching at 2948.6 cm−1, C=O stretching at 1748.2 cm−1 and C-0=0 stretching at 1181.2 cm−1 and 1038.8 cm−1, which is a characteristic ester bond. Bending vibrations for CH3 asymmetric and CH3 symmetric were identified at 1453.1 cm−1 and 1381.7 cm−1, respectively [−1 which can not be seen in the core-shell structured nanofibers. This indicates that the PVA is not on the surface of the fibers, which is an additional proof of effective encapsulation in core-shell nanofibers [−1 were observed in the nanofibers and FTIR spectra of MH nanofiber are shown in FTIR spectroscopic imaging in ATR  mode permits investigation of the molecular and structural properties of polymers . FTIR spectively ,33. PVA nofibers . After tAs a potential wound dressing material, composite nanofibers should promote cell growth and cell differentiation. The in vitro cytotoxicity tests of coaxial nanofibers toward HaCat cells were evaluated using MTT assays. As shown in y = 0.1178x−0.0083 (R = 0.9898) where y is the MH concentration (in micrograms per milliliter) and x is the solution absorbance at 233 nm which is MH UV absorbance peak. The amount of MH released from the fibers was determined by UV spectroscopy using a pre-determined calibration curve, 2R) of all samples are given in 2R values for all samples than other kinetics. Furthermore, in the Korsmeyer–Peppas model, the value of n describes the mechanism of drug release from a polymeric system. For a cylindrical system, 0.45 ≥ n corresponds to a Fickian diffusion and 0.45 < n < 1 refers the Non-Fickian diffusion [n values of nanofiber samples are less than 0.45, suggesting MH release from nanofiber samples follow the typical Fickian diffusion mechanism.The release kinetics of MH drug was investigated by fitting with various mathematical models such as zero order, first order, Higuchi and Korsmeyer–Peppas models. The obtained kinetic constants and regression coefficients (iffusion . As showm) of the nanofibers. Neat- and drug-loaded nanofibers showed double peaks at the melting point, the first one at about 163 °C and the second one at 165 °C. According to previous studies, it seems possible that these results are due to the formation of imperfect crystals during the electrospinning process [Differential Scanning Calorimetry (DSC) has been widely used to investigate the crystal structures of electrospun nanofiber mats. It is important to understand the possible structural changes of the biomaterial, such as the change in melting temperature, degree of crystallinity, and enthalpy. The DSC thermograms of the Neat, MH, MHFSP1, MHFSP3 and MHFSP5 coaxial nanofibers are presented in  process . DSC the process  and also process .2R values for all the samples than other kinetics. Indeed, release kinetics of the nanofibers were described on the typical Fickian diffusion mechanism according to the Peppas–Korsemeyer kinetic. Overall, this study reveals that the composite nanofibers had a good potential to be used in the treatment of diabetic ulcers.In this study, drug and protein loaded nanofibers were successfully fabricated by coaxial electrospinning. Fish Sarcoplasmic Protein (FSP) was produced from Atlantic Bonito and incorported into core polymeric matric at different concentrations. The morphology and chemical compositions of the nanofiber samples were observed using a Scanning Electron Microscopy (SEM) and Fourier Transform Infrared Spectrometer (FTIR). Furthermore, Differential scanning calorimetry (DSC) studies were carried out to investigate the thermal characteristics of the nanofiber mats. According to the results, the coaxial nanofiber mats showed uniform bead-free fiber morphology at an average 650 nm and presented characteristic peaks of the content in the FTIR study. DSC results indicate that the crystallinity of the coaxial nanofibers was reduced with the presence of FSP. The coaxial nanofibers also showed suitable properties for proliferation, and attachment of HaCaT cell lines. Drug release from the coaxial nanofibers were followed by UV spectroscopy and the release profiles were fitted to four popular mathematical equationsm such as zero order, first order, Higuchi and Peppas–Korsemeyer equations to understand the kinetics of drug release. The first order kinetic have better agreement with higher


Desmogleins (Dsg2) are the major components of desmosomes. Dsg2 has five extracellular tandem cadherin domains (EC1-EC5) for cell-cell interaction. We had previously confirmed the Dsg2 antibody and its epitope (named KC21) derived from EC2 domain suppressing epithelial-mesenchymal transition and invasion in human cancer cell lines. Here, we screened six peptide fragments derived from EC2 domain and found that KR20, the parental peptide of KC21, was the most potent one on suppressing endothelial colony-forming cell (ECFC) tube-like structure formation. KC21 peptide also attenuated migration but did not disrupt viability and proliferation of ECFCs, consistent with the function to inhibit VEGF-mediated activation of p38 MAPK but not AKT and ERK. Animal studies showed that KC21 peptides suppressed capillary growth in Matrigel implant assay and inhibited oxygen-induced retinal neovascularization. The effects were comparable to bevacizumab (Bev). In conclusion, KC21 peptide is an angiogenic inhibitor potentially useful for treating angiogenesis-related diseases.The online version of this article (10.1007/s12265-019-09865-6) contains supplementary material, which is available to authorized users. Desmosomes provide strong adhesion to maintain tissue function and organ architecture. Organs that frequently experience mechanical stress, such as the skin and heart, particularly express abundant desmosomes to provide plasma membrane attachment sites for adjacent cells . DesmosoThe therapeutic potential of endothelial progenitor cells (EPCs) has gained great interest since the observations that a significant number decrease of circulating EPCs was detected in patients with severe conditions, such as diabetes and repeated hospitalization for heart attacks . EPCs isPreviously, we had identified the antagonist role of Dsg2 on cancer metastasis . Polyclo+KDR+AC133+CD31+ as described [4 cells/cm2 were seeded on 1% gelatin-coated dish (BD Biosciences) and maintained in the 37 °C incubator under a humidified 95% air and 5% CO2 atmosphere.Ethical approval (No. 15MMHIS112) was granted by the Mackay Memorial Hospital Institutional Review Board, Taipei, Taiwan. Informed consent was obtained from healthy donors before the collection of peripheral blood (80 mL). The peripheral blood mononuclear cells (PBMCs) obtained from healthy donors were fractionated from other blood components by centrifugation. EPCs were isolated using CD34 MicroBead kit and MACS cell separation system (Miltenyi Biotec). In this study, PBMCs were cultured for 28 days to get ECFCs (late ECFCs) as described . ECFCs wCell viability was measured using the cell counting kit-8 (CCK-8) (Sigma-Aldrich) to reflect the dehydrogenase activity of living cells. ECFCs were seeded onto 96-well plates and treated with Dsg2-derived peptides . Twenty-four hours later, CCK-8 solutions were added to each well for 4 h, and the medium was harvested for the measurement of absorbance at 450 nm using a microplate reader. For cell proliferation assay, ECFCs were treated with Dsg2-derived peptide  for 4 h and then fixed. Cells labeled with 5-bromo-2′-deoxyuridine (BrdU) were subsequently identified with a primary antibody against BrdU and visualized with a secondary antibody conjugated with horseradish peroxidase using tetramethylbenzidine as a substrate.Immunostaining of cell cultures was described previously . AntibodECFCs were seeded at 80% confluence on 60-mm dishes in MV2 complete medium. Next day, cells were incubated in MV2 medium with 2% FBS with various concentrations of KC21 peptides for 24 h. Fifty μg of the conditioned medium was analyzed by 10% zymogram gel containing 0.1% gelatin . After eECFCs were treated with KC21 or scramble peptides for 24 h. Conditioned media were harvested for PAI-1 activity determination. PAI activity assay kit (Chemicon) utilized a chromogenic substrate cleaved by active uPA and detected by its optical density at 405 nm. Addition of PAI-1 in conditioned media blocked the cleavage of substrate by uPA. The relative PAI-1 activity was obtained by plotting with the standard curve of 10 units of uPA inhibited by a series dilution of PAI-1 incubated at 37 °C for 2 h as the assay instructions described.ECFCs were lysed with SB-20 buffer , and protein concentrations were determined by modified Lowry’s method. Aliquots of cell lysates were loaded into 10% SDS-polyacrylamide gels, electrophoresed, and transblotted onto polyvinylidene fluoride membranes (Millipore). The blots were blocked with 10% bovine serum albumin for 1 h and probed with indicated primary antibody for two hours. The blots were further incubated with alkaline phosphatase-conjugated secondary antibodies for 1 hour at a room temperature. Immunoreactivity was visualized using CDP-star system (Roche) according to the manufacturer’s instruction. Primary antibody for Dsg2 (Santa Cruz), p38, p-p38, Akt, p-Akt, ERK, p-ERK, MMP9, and PAI-1  were diluted with PBS in 1 to 1000.ECFCs were grown on twenty-four-well plates to reach confluence. Cell-free gap was generated using SPLScar™ Block  and photographed by optical microscopy  at × 40 magnification as basal line. After 4-h culture, cells were fixed and imagined to measure new growth areas using Image-J software (NIH). The ratio of the new migration area was calculated relative to the initial wound area and normalized to that for PBS-treated cells as described [4 cells in MV2 medium containing 2% FBS for 24 h at 37 °C in a 5% CO2 humidified incubator. Each sample was tested in triplicate on the same plate, and wells were photographed with a Leica microscope with camera (× 40 magnification). Five fields were randomly chosen in each well to measure tube length and junction number manually using Image-Pro Plus 6.0 . Total tube length and junction number per field were calculated.Growth factor-reduced Matrigel (BD Biosciences) was thawed at 4 °C before use. Twenty-four-well plates were coated with Matrigel (200 μL/well) and polymerized for 30 min at 37 °C. ECFCs resuspended with various concentrations of KC21 peptides, scramble peptides, and antiDsg2 antibody (10 ng/ml or bev (0.25 μg/mL)) were seeded on Matrigel-coated wells at a density of 5 × 10All animal experiments were approved by the Institutional Animal Care and Use Committee of the Mackay Memorial Hospital. C57BL/6 mice were kept and bred in accordance with the institutional ethical committee guidance .To assess the antiangiogenic effects of KC21 peptides in vivo, growth factor-reduced liquid Matrigel (0.5 mL) containing heparin (60 U/mL), VEGF , and KC21 peptides or scramble peptides were subcutaneously injected into the mice near the abdominal midline. Seven days after injection, mice were euthanized and Matrigel plugs were surgically removed. For macroscopic analysis of angiogenesis, hemoglobin content in Matrigel was measured with Drabkin’s reagent kit 525 (Sigma-Aldrich).Retinal neovascularization was induced by the use of a well-established murine model of oxygen-induced retinopathy . Neonata+KDR+AC133+CD31+ [We had identified the antagonist role of EC2 domain in suppressing EMT and invasion of human cancer cells  . In thi33+CD31+ . The fraDsg2 is an integral membrane protein with five tandem cadherin domains EC1-EC5) . The app-EC5 . ThAt first, we measured the effects of KC21 peptides on inhibiting ECFC angiogenic potential, showing in a dose-dependent manner. KC21 is with similar potency to its parental peptide KR20 Fig. . EffectsWe used Madin-Darby Canine Kidney (MDCK) cells, an epithelial cell line well known for the expression of functional desmosomes, to detect Dsg2 expression pattern. As shown in Fig. To test whether KC21 interacts with Dsg2 in vivo, the N-terminus of KC21 was conjugated with FITC  to track its cellular distribution. FITC-KC21 was taken up by MDCK cells within 30 min after treatment Fig. .As KC21 peptides show an antiangiogenic potential, we next asked whether KC21 inhibits ECFC migration, the prerequisite step of angiogenesis. As shown in Fig. VEGF is a major angiogenic factor in regulating PI3K/Akt for endothelial cell survival, PLC-γ/ERK for proliferation and p38 MAPK for cell migration –29. KC21Since KC21 inhibits ECFC tube-like structure formation in vitro, we tested whether it inhibits VEGF-induced angiogenesis in vivo by subcutaneous Matrigel plugs assay. VEGF (10 ng/mL) profoundly induced capillary growth in plugs containing PBS and scramble peptides, while the induction was significantly abolished by KC21 peptides Fig. a. The coDigestion of the extracellular matrix is the initial step of angiogenesis. Therefore, we further tested the effects of KC21 on angiogenic MMPs in ECFC culture. The results showed that KC21 peptides specifically inhibit the activities of extracellular MMP9, but not MMP2 or MMP3 Fig. c, e. TheIt has been proposed that activation of MMP9 is regulated by plasminogen/plasmin fibrinolytic system, controlled by plasminogen activator inhibitor-1 (PAI-1) . We testIn order to evaluate the antiangiogenic effect of KC21 on pathogenic neovascularization, we compared the treatments of KC21 with Bev, an FDA-approved therapeutic antibody for AMD. As shown in Fig. Our study showed that the designed KC21 peptides have the ability to inhibit human ECFC migration in vitro, to reduce VEGF-induced capillary growth and to suppress oxygen-induced retinal neovascularization in vivo. At the cellular level, KC21 peptides specifically attenuate VEGF-induced activation of p38 MAPK, but not the signaling targets of Akt and ERK. Also, KC21 peptides regulate MMP9 and PAI-1 to stabilize the extracellular matrix of ECFCs.The functions of Dsg2 on angiogenesis have gained attention recently. Depletion of Dsg2 by siRNA impaired tube-like structure formation in MVECs . These fProteolysis of extracellular matrix is an initiation step for the recruitment of endothelial progenitor cells to establish new capillaries. Matrix metalloproteinases (MMPs) are extracellular endopeptidases selectively degrading components of the extracellular matrix. Shedding Dsg2 ectodomains by MMPs had been detected in the inflamed intestinal mucosa of mice with colitis and patients with ulcerative colitis . In thisThe therapeutic functions of mature endothelial cells on ischemic diseases have been tested in hindlimb ischemic animal model . Both KCTo our knowledge, this study is the first report showing evidences for Dsg2-derived peptides to suppress retinal neovascularization in the mouse oxygen-induced-retinopathy (OIR) model. Neovascularization causes ocular vessel leakiness, edema, retinal detachment, and even blindness. VEGF is a major hypoxia-induced angiogenic factor and is found to be increased in the vitreous and retina to exacerbate retinopathy. Clinically, therapeutic agents against VEGF, such as bev and aflibercept, have been widely used to control the overgrowth of retinal blood vessels for vision rescue. However, more than 30% of the patients do not respond to these therapies and adverse events were also reported . The finESM 1Phenotypic characterization of human ECFCs and effects of KC21 peptides on ECFC and endothelial cell angiogenesis (DOCX 1395 kb)


Mycobacterium bovis, can affect domestic and wild animals as well as humans. Identifying the major transmission mechanisms in an area is necessary for disease control and management. In this study, we aimed to evaluate the involvement of different types of contact in M. bovis transmission between cattle farms of south-western France between 2007 and 2015. We analyzed an empirical contact network of cattle farms as nodes, with known infection status and molecular types (16 circulated during the study period of which 14 affected only cattle and two both badgers and cattle). Edges were based on cattle trade data (T-edges) and on spatial neighborhood relationships between farms, either direct (P-edges) or badger-mediated, when two farms neighbored the same badger home range (B-edges), or two distinct but neighboring badger home ranges (D-edges). Edge types were aggregated so that the contact network contained only unique edges labeled by one or several edge types. The association between the contact network structure and bTB infection status was assessed using a non-parametric test, each molecular type being considered a marker of an independent epidemic. Using a logistic regression model, we estimated the contribution of each edge type to the probability for an edge originating from an infected farm to end at another infected farm. A total number of 1946 cattle farms were included in the study and were linked by 54,243 edges. Within this contact network, infected farms (whatever the molecular type) always belonged to the same component, suggesting the contact network may have supported bTB spread among those farms. A significant association between the pattern of bTB-infected farms and the structure of the contact network was observed when all the molecular types were simultaneously considered. The logistic regression model showed a significant association between M. bovis infection in direct neighbors of infected farms and the connection by T-, B- and D-edges, with odds-ratios of 7.4, 1.9, and 10.4, respectively. These results indicate a multifactorial M. bovis transmission between cattle farms of the studied area, with varying implication levels of the trade, pasture and badger networks according to the molecular type.Bovine tuberculosis (bTB), mainly caused by  Mycobacterium bovis, the main agent of bovine tuberculosis (bTB) has been found in a wide variety of domestic and wild animal hosts, as well as in humans (M. bovis is cattle (Cervus elaphus) (Capreolus capreolus) (Vulpes vulpes) (Sus scrofa) (Meles meles)  , 11, roepreolus) , red fox vulpes) –16, wild scrofa) , 18 and s meles) –21.M. bovis transmission between wild and domestic hosts. The largest part of M. bovis shedding seems to occur through aerosols (respiratory tract secretions) and to a lesser extent through saliva, urine, feces  that foster M. bovis transmission  contacts due to the trade of live cattle  of 2013 provided by the French Ministry of Agriculture. Two pastures were considered neighbors if the minimal distance between their borders was less than 3 m. Farm sizes  and types  were obtained from the French cattle tracing system  (Table The study population was made up of the 1946 farms having reported cattle between January 2007 and March 2016 (end of the 2015 herd skin-testing period) and owning at least one pasture included in a 2,735 km2 study area, an area straddling the border of Landes department, and triennial in Pyrénées-Atlantiques department. M. bovis infection was confirmed by polymerase chain reaction (PCR) and/or bacterial culture (either following a positive skin test or the detection of a suspect lesion during routine meat inspection at a slaughterhouse)  where infected farms had been detected the previous year, as well as in the neighboring ones, using either single intradermal comparative tuberculin tests (SICTT)  or single intradermal tuberculin tests (SITT) , both performed in the cervical region. In the other communes of the study area, herd testing was biennial in erhouse)  in 69 caerhouse) . A uniquM. bovis earlier in a given farm than in another one does not imply that the former had been infected earlier than the latter. For this reason, the detection dates could not be taken into account.A farm was classified infected by a given molecular type if this type had been detected at least once in the farm during the study period. Because of the geographic differences in the frequency of skin testing, having detected Two thousand four hundred and 25 badger setts were identified and geolocalised by hunters in the study area, between 2013 and 2015. Around those setts, considered as main setts , we defined badger home ranges using a two-step procedure: (i) a Dirichlet tessellation was first built around all setts  and ii. Two sethttps://www.plateforme-esa.fr/filedepot_download/35377/100) and snares were checked the morning after the day they were set up within the 2 h following sunrise, in order to limit the stress of trapped badgers. Trapped badgers were culled by head shot except in a minority of cases where they were found already dead . Road-killed badgers were also considered. Stopped restraints used for trapping were placed near sett entrances, those setts being considered as the sett of the trapped animals. Where badgers were found dead along roads, hunters reported the most probable sett according to their knowledge of the area , 11.2% were detected infected , of which 39 harbored the SB0821 molecular type and 6 the SB0832 molecular type, both molecular types having also been found in cattle (Table n = 75). All the remaining setts, either with only one badger tested negative or without analyzed badger were considered of unknown status.BTB surveillance data were provided by the French Ministry of Agriculture. In the study area, bTB surveillance in badgers was performed according to the “Sylvatub” surveillance network, which started in 2012 in the study area . Surveilthe area . All theA contact network was built using farms of the study population as nodes, and four types of edges Figure :i to farm j represented the sale of one or several cattle by farm i to farm j during the study period, at one or several occasions;- A trade edge (denoted T-edge below) from farms i and j represented the fact that a pasture owned by i and another one owned by j were neighbors;- A pasture neighborhood edge (denoted P-edge below) between farms i and j represented the fact that both farms were neighbors of a given sett;- A simple badger-mediated edge (denoted B-edge below) between farms i and j represented the fact that (i) farm i was neighbor of a sett k1, (ii) farm j was neighbor of a sett k2, and (iii) the setts k1 and k2 were themselves neighbors.- A second level badger-mediated edge (denoted D-edge below) between farms To avoid duplicated edges, the types of edges  were aggregated at the edge level. The full contact network thus contained only unique edges labeled by one or several edge types Table . BecauseSubnetworks were extracted from the full contact network by restricting the edges to those of specific types Table . These s- the cattle-specific network incorporated edges labeled T, P or T-P, thus representing only contacts induced by cattle breeding practices;- the badger-specific network incorporated edges labeled B, D or B-D, thus representing only badger-mediated contacts;- the mixed network incorporated all the remaining edges, thus representing the co-occurrence of cattle-specific and badger-mediated contacts.M. bovis identified in the study area was considered as a marker of an independent epidemic. For a given molecular type, the contact network may be considered as supporting M. bovis transmission between two farms only if a path exists in the network between these farms. The transmission tree rooted on a detected infected farm should then be entirely located in a single component of the network. The contact network may then be considered as supporting the spread of a given molecular type if most of the farms infected by this molecular type are located in the same component of the contact network. We thus first computed, for each molecular type identified in more than one farm, the number of components in which these infected farms were located . The observed value of this statistic is then compared to the distribution of the same statistic obtained by randomly reallocating the location of cases, thus simulating a possible pattern of cases under the null hypothesis of an absence of association between bTB case location and network structure. The empirical p-value of the k-test is then the proportion of permutations for which the k-statistic is greater than the observed one. We adapted this test to a multi-type epidemic by redefining the k-statistic as the mean number of cases among the neighbors of a node, which were infected by the same molecular type as that node.To evaluate whether the observed pattern of bTB infected farms may have resulted from transmission processes in the contact network, we used the l et al. . This pek-test was first performed on the full contact network. It was then applied on the cattle-specific, badger-specific and mixed subnetworks; and this, for two groups of molecular types: those observed in cattle only and those observed in cattle and in badgers. Seven tests were thus performed and the Bonferroni correction was applied. Ten thousand permutations were used to compute the empirical p-value.The To further analyse the association between edge types and bTB occurrence, we focused on edges originating from infected farms. A binary status was assigned to each of these edges, with a value of 1 when the destination node was infected by the same molecular type as the originating node, and 0 otherwise. The association between this status and the edge type was then assessed using a case-control design: cases were edges having a status of 1, and controls the edges having the status 0. Four binary explicative variables were defined, based on the types labeling the edge: T, P, B, and D. In addition, we took into account the size  of the edge originating and destination farms, herd size being a well-known risk factor for bTB detection in cattle farms . We thusThe definition of badger-mediated edges was based upon the neighborhood between pastures and one (B-edges) or two (D-edges) badger home ranges. For some of the corresponding setts, the trapping results allowed defining an infection status: setts were considered as (i) infected when at least one trapped badger had been found infected with an identified molecular type and (ii) uninfected when at least two trapped badgers had been tested negative and no occupant badger had been found infected . Based Dirichlet tessellations were computed using the deldir package  and buffWithin the full contact network, the most frequent edge type was the combination of B- and D-edges, followed by single D-, T-, and B-edges. The P-edge type was less frequent alone than in combination with the other types Figure .The largest weak component of the full contact network incorporated 99.8% of the study population. Regarding the four edge-type-specific networks, the proportion of nodes included in the largest component was higher in trade and badger related networks  than in the pasture network (50.4%) Table , 2.For each of the 16 molecular types, the farms where the type had been observed were always located in the same component of the full contact network. This was also the case for the B-network, but not for the T-, P-, and D- networks Table .M. bovis transmission between an infected farm and its direct neighbors in the full contact network.Four molecular types were observed in at least two detected infected farms Table . For 87%p = 0.13).We computed the proportion of shortest paths made of a single edge between farms infected (i) by molecular types found only in cattle and (ii) by molecular types found both in badgers and cattle. The difference between these two proportions was not significant  . Finally, the structure of the mixed network was significantly associated with the pattern of bTB-infected farms for both groups of molecular types (p = 0.006 and p < 7.14*10−3 respectively) (Table Using ) Figure . No signThe four edge types were included in the logistic regression model as no significant multicollinearity was detected. T-, B-, and D-edge types were significantly associated to the probability of being a case with an OR of 7.13 for the T-edge type (95% CI: [3.39–15.06]), 1.89 for the B-edge type (95% CI: [1.32–2.76]) and 10.44 for the D-edge type (95% IC: [4.38–26.66]). The size of the destination farm of the edge was also significantly associated to the probability of being a case. Regarding edge types, attributable risk fractions were 84% for the D edge type, 32% for the B edge type, and 12% for the T edge type (Table p < 0.0001) with an associated OR of 7.3 [95% CI: (2.9–21.9)].Among edges representing badger-mediated transmission , the infection status of badger setts involved (one sett regarding B-edges and at least one of the two setts regarding D-edges) was known for 264 edges (5%) originating from a farm infected by one of the two molecular types shared between badgers and cattle. Among them, 44 were case edges  of which 38 (86%) were supported by positive badger setts; and 220 were control edges of which 102 were supported by positive badger setts (46%). These differences were significant  to a herd detected infected were considered having been submitted to similar testing regimens, both for B and D edge types (as in most cases the connected farms were located in the same commune), and for the T and P edge types (because of contact tracing). As only edges originating from herds detected infected were considered in the Taking into account the molecular types of isolates allowed considering 16 independent epidemics, of which 12 appeared restricted to a single farm, and 14 to less than 10 farms. All of these 14 molecular types affected only cattle. This predominance of molecular types found in a single cattle farm (75%) was in line with a previous study carried out in France between 1979 and 2000 in which a large majority of molecular types (84%) were found at a low frequency (less than 10 farms). This result has been interpreted as the sign of a poor spread of these strains , which cM. bovis infection in the study area. This result is in line with multifactorial mechanisms of bTB spread previously suggested by other studies , the strong connectivity resulting from the union of the four networks into the full contact network provided a structure that might enable the spread of the  studies , 29. As n routes . Prominek-tests, a significant association was observed between the pattern of bTB-infected farms and the structure of the full contact network. Moreover, the structures of the badger-specific and mixed networks were significantly associated with the pattern of farms detected infected by molecular types shared between badgers and cattle. This result was expected and confirmed that badger-mediated edges could be viewed as paths for the interspecies M. bovis transmission. In addition, the structure of the mixed network was significantly associated with the pattern of bTB-infected farms for molecular types found only in cattle, whereas it was not the case for the cattle-specific network. We could assume that the spread of cattle molecular types would be more efficient when direct contact (trade and/or pasture neighborhood) are associated with indirect badger-mediated contacts. In addition, we should be cautious about the cattle specificity of these molecular types, as these molecular types may be (or have been) present in the badger population without being observed, because of the relatively low sensitivity of bTB surveillance in the badger population.Using M. bovis through the edge, although the edges of the full contact network represent possible transmission paths for the bacteria and case edges thus represent possible transmission events. The largest odds-ratio was attributed to the D edge type, followed by the T edge type. This predominance of badger-mediated edges reflects the specific situation of the study area, where molecular types shared between badgers and cattle were predominant  or at the originating farm only (control edges). Because the detection dates could not be considered in the study to infer dates of infection, the co-occurrence of the same molecular type at both ends of case edges does not model the transmission of M. bovis (AF = 12%). This result is in agreement with a previous French study conducted at the national scale, according to which the population attributable risk fraction of bTB infection had been estimated at 12% [5–18%] for cattle trade . This short distance does not allow other opportunities of direct contacts between cattle, such as the wandering of livestock, to be represented.In a previous study conducted in France, pasture neighborhood was found significantly associated with the farm infection status . HoweverOR = 7.3; 95% CI:[2.9-21.9]). This result supports an actual badger-mediated transmission through these types of edges. Nevertheless, wild boars have also been found infected with M. bovis within the study area. Indeed, among 548 analyzed wild boars between 2011 and 2015, 15 (2.7%) were found infected. The corresponding molecular types found in these wild boars were the two molecular types shared between badgers and cattle. Therefore we cannot exclude the role of this wild species that we could not consider in this study because of a lack of field data that would have allowed its spatial organization  to be represented. Not considering wild boars in our analyses could have led to an over-estimate of the role of B and D edge types in M. bovis transmission between cattle farms.The badger-specific edges (B and D edge types) were defined based on sett locations, one or two setts being associated to each. For some of these setts, an infection status could be determined based on bTB surveillance data. We showed that this infection status was significantly associated with the fact that the sett as well as the originating and the destination farms had all been found infected by isolates of the same molecular type (Other indirect contacts through herd practices could also have contributed to the predominance of the D edge type. Indeed, this type of edge created links between farms without direct contacts at pasture but being in a kind of vicinity. As examples, the sharing of material or the loan of animals could create links between farms that may overlap the D edges. However, no data were available to investigate this assumption. Its confirmation or refutation would require supplementary investigation.M. bovis transmission between cattle farms within the Pyrénées-Atlantiques–Landes area, France from 2007 to 2015. The largest part of bTB spread seemed to be due to badger-mediated contacts, however cattle trade played a significant role. Consequently, to be truly effective, control measures should not focus on a single type of contact but ought to act on the different mechanisms we raised.In conclusion, this study supports the multifactorial nature of MB-Z, AC, and BD conceived and designed the study. MB-Z prepared the data for the analysis. MB-Z and BD performed the analysis. MB-Z wrote the manuscript. MB-Z, AC, and BD revised the manuscript. All the authors approved the submitted version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sir,et al.In an interesting contribution, Limmathurotsakul 2 equivalent. Going beyond the greenhouse gas metric alone, another eye-catching interpretation suggests that humanity uses the equivalent of 1.7 Earths to provide the resources we use and absorb our waste. In other words, the globe has an ecological deficit and the calculation can even pinpoint the day of the year when this starts.However, it is important to be clear on what a footprint concept represents and to consider that its application to antibiotics may potentially be misleading. Originally, the ecological footprint signifies the resource requirement to offset ecological damage,et al.,Footprints provide a compelling message to governments and consumers about the sustainability or otherwise of current consumption trends (e.g. there is no planet B) and are a precursor to literature on planetary boundaries.There are several potential policy-relevant metrics to represent antibiotic use and it is important to be clear on their interpretation in the AMR context. We should also avoid overstating the power of footprinting rhetoric, when evaluation evidence is conspicuously absent. More importantly, apart from drug innovation, there is no obvious offsetting analogy applicable to AMR. It is important that this message is clear in any metric.The author acknowledges support from UK Newton Fund awards co-funded by the Economic and Social Research Council grant numbers ES/S000186/1 and ES/S000208/1, linked to the India Department of Biotechnology grant numbers BT/IN/Indo-UK/AMR/06/BRS/2018-19 and BT/IN/Indo-UK/AMR/03/RKE/2018-19.None to declare.