Several Genome Wide Association Studies (GWAS) have reported variants associated to immune diseases. However, the identified variants are rarely the drivers of the associations and the molecular mechanisms behind the genetic contributions remain poorly understood. ChIP-seq data for TFs and histone modifications provide snapshots of protein-DNA interactions allowing the identification of heterozygous SNPs showing significant allele specific signals (AS-SNPs). AS-SNPs can change a TF binding site resulting in altered gene regulation and are primary candidates to explain associations observed in GWAS and expression studies. We identified 17,293 unique AS-SNPs across 7 lymphoblastoid cell lines. In this set of cell lines we interrogated 85% of common genetic variants in the population for potential regulatory effect and we identified 237 AS-SNPs associated to immune GWAS traits and 714 to gene expression in B cells. To elucidate possible regulatory mechanisms we integrated long-range 3D interactions data to identify putative target genes and motif predictions to identify TFs whose binding may be affected by AS-SNPs yielding a collection of 173 AS-SNPs associated to gene expression and 60 to B cell related traits. We present a systems strategy to find functional gene regulatory variants, the TFs that bind differentially between alleles and novel strategies to detect the regulated genes. In addition, since the target genes are often not obvious from the associations, most of the molecular mechanisms behind the genetic contributions to immune and autoimmune diseases still remain poorly understood.More than 15% of the variants reported today in the Genome Wide Association Studies (GWAS) catalog are associated to immune system diseases. It is today established that the top hits in GWAS rarely drive the associations2.In recent years, the study and better understanding of the complexity of autoimmune diseases has prompted a shift from an almost exclusively T cell mediated view to a more synergistic view with a prominent role for B cells. Several functions mediated by B cells, such as secretion of autoantibodies, inflammatory cytokines, presentation of autoantigens, modulation of antigen processing etc., are today consistently reported as central in the onset of several autoimmune diseases3 are nowadays gaining a prominent role in explain the etiology of systemic lupus erythematosus (SLE) characterized by the production of antinuclear antibodies; rheumatoid arthritis (RA) a chronic inflammation of the joint capsule and synovial membrane; multiple sclerosis (MS) characterized by multifocal inflammation, demyelination, gliosis and axonal loss in the central nervous system (CNS); inflammatory bowel disease (IBD), a chronic relapsing intestinal inflammatory disease classified into two major forms, Crohn’s disease (CD) and ulcerative colitis (UC); type 1 diabetes (T1D), an autoimmune disease in which insulin-producing β-cells in the pancreatic islets are destroyed and many more autoimmune, allergic and socially impairing diseases .Regulatory B cells4. Therefore, linking genomic variation to diseases or phenotype is a complex process that involves three major steps: (i) identify the causal gene regulatory variant(s), (ii) identify the TF(s) that bind to the variants, (iii) identify the target gene(s) whose deregulation lead to the phenotype. This opens the field for functional studies of the biological mechanisms of disease.The majority of genes show difference in activity between people and it has been proposed that a majority of drivers of GWAS signals are located in non-coding regulatory elements and affect the binding of transcription factors (TFs) leading to allelic difference in expressionThe majority of the GWAS top associated variants are located in non-coding regions and often in high linkage disequilibrium (LD) with several other variants making it difficult to pinpoint the real functional SNP(s). One way to find putative functional variants is to detect regions with allele specific (AS) binding of TFs or their surrogates histone modifications, suggesting a different regulatory downstream role based on the individual genotypes. A powerful way to do this is to study the heterozygous positions in a cell/tissue so that one allele acts as the internal control for the other. A limitation is that when studying one person you only survey some common variants and a small fraction of all rare ones. However, if you study the same cell/tissue in several individuals you cover a larger fraction of common variants, which are the relevant ones for GWAS studies. ChIP-seq data for TFs and histone modifications provide snapshots of direct and indirect protein-DNA interactions allowing the identification of heterozygous SNPs with significant allele-specific signals (AS-SNPs).5 and ChIP-seq6, this concept has been exploited by several bioinformatics approaches such as sTRAP7, rSNP MAPPER8, AlleleSeq9, iASeq10 and ALEA11 that have been developed to use RNA-seq and ChIP-seq datasets in order to gather information about allele specific expression (ASE) and binding (ASB). Allele specific variants have also been detected using data from DNaseI hypersentitive sites (DHSs) analyses12.The central idea is that a regulatory functional variant can change a TF binding site resulting in altered gene regulation. After our original discovery that AS signals can be detected using ChIP6 that interrogating ASB events in one cell line is limited to the 33% heterozygous common SNPs defined by the Hardy-Weinberg equilibrium. Moreover, we demonstrated how a small set of ChIP-seq experiments for TFs allows the detection of most common AS-SNPs. This approach aimed at prioritizing candidate functional variants has proven quite robust with up to 70% of the AS-SNPs tested via luciferase assays in HepG2 cells showing an allele specific enhancer activity13. Here we present the results of the identification of SNPs that show allele-specific behavior using a minimal set of ChIP-seq datasets produced for three histone modifications and two genome architectural proteins in a population of seven B cell lines where the likelihood of finding heterozygous variants with an allele frequency (AF) of 0.35–0.65 in at least one individual is 99%, greatly increasing the pool of heterozygous common SNPs that can be investigated for AS signals. In this set of cell lines we interrogate 85% of common genetic variants in the population for potential regulatory effect. Increasing the population size is also beneficial toward the identification of rare AS-SNPs, but the huge number of rare variants in the human population means that only a small fraction of them is heterozygous when analyzing seven individuals.We have previously shown14, JASPAR15, UniPROBE16 or HOCOMOCO17 that provide experimentally proven binding sites (BSs) and motif models typically represented by positional weight matrices (PWMs). DNA sequences of interest are then matched against these PWMs to predict which TF may putatively bind the selected sequence. One limitation of PWM models is that they neglect intra-motif dependencies, so motif models that take into account intra-motif dependencies such as Markov Models19, Transcription Factor Flexible Models (TFFMs)20, Variable-Order Markov Models (VOMMs)21, or Parsimonious Markov Models (PMMs)22 have been developed for an improved prediction of BSs. These motif models take into account intra-motif dependencies by considering conditional probabilities where the probability of the nucleotide at the current motif position may depend on the previous k nucleotides. For traditional Markov Models, the number of parameters at each motif position increases exponentially with the order k, which may lead to overfitting and poor prediction. In contrast, TFFMs, VOMMs, and PMMs address this problem by a variable choice of subsets of nucleotides considered as conditioning variables in a position-specific manner and have been shown to outperform PWM models in the context of motif discovery24. However, these motif models do not lead to an improved prediction of BSs for every TF, but one noteworthy feature of these motif models is that in the presence of intra-motif dependencies the improvement is typically substantial, whereas in the absence of intra-motif dependencies the decrease is typically minor or not measurable, so that utilizing these motif models, as in the present study, can be said to be often potentially beneficial and almost never potentially harmful24.One traditional approach to identify the DNA-binding TF is to utilize databases such as TRANSFAC25, variants are also located in intergenic cis-acting enhancer regions that can act at a distance of tens or hundreds of kb, precluding an easy prediction of the target gene(s). Promoter-enhancer interactions are often restricted to specific regions by the TF CTCF, which together with SA.1 and other proteins in the cohesin complex creates topologically associated domains (TADs). Allele specific behavior of CTCF and SA.1 may change domain size and thereby different promoter-enhancer interactions may occur between alleles26. Several techniques have been developed to study promoter-enhancer interactions on a genome-wide scale. Hi-C was the founder technique to define TADs and higher-order chromatin interactions genome-wide27. Based on TADs and long-range interaction data it is possible to move past the prediction that the closest gene is the likely target since it is relevant to consider the genes located in the TADs where the candidate regulatory variants are located. Targeted chromosome conformation capture  is a novel technique evolved from Hi-C yielding promoter-enhancer interactions to a much higher resolution29. A single HiCap experiment can employ approximately 40,000 capturing probes targeting gene promoters annotated in RefSeq database to provide a genome wide promoter-enhancer interaction network.Finally, the question remains on how to identify the target gene(s) controlled by the regulatory element. Historically the closest gene to a GWAS SNP was considered the likely candidate. While this holds true in many casesIn this study we address the three steps presenting a systems strategy to find functional gene regulatory variants, the TFs that bind differentially between alleles and novel strategies to detect the regulated genes. We identified 17,293 unique AS-SNPs across 7 lymphoblastoid cell lines. To elucidate possible regulatory mechanisms we evaluated their associations to immune GWAS traits and to gene expression in B cells. Lastly, we integrated long-range 3D interactions data to identify putative target genes and motif predictions to identify TFs whose binding may be affected by AS-SNPs.30 were then aligned to the respective genomes to identify heterozygous sites with allele specific signals and the results were corrected for genome-wide testing and pruned for false positives by filtering out AS-SNPs in CNVs and different types of repeated regions (see Methods). The cell specific collections of AS-SNPs obtained in this way were then intersected with GWAS and eQTL SNPs identified in B cells in order to link the allele-specific behavior observed in ChIP-seq to B cell specific traits or gene expression. Finally, in order to gather more information about the effect of the AS signals, we used long-range interaction data to prioritize the targets of the identified regulatory SNPs and motif analysis to identify candidate TFs mediating the regulatory process.We used ChIP-seq data and genomic sequences from seven B cell lines to search for AS-SNPs. Initially the two personal genomes were created by replacing the reference bases at variable sites with the bases supplied by a cell-specific SNPs collection (see Methods). The ChIP-seq reads from histone modifications defining promoters (H3K4me3), enhancers  and domain boundaries proteins To ensure that the AS-SNPs identified from histone modifications ChIP-seq data were due to the AS activity of the regulatory element we selected only AS-SNPs within the regulatory element whose boundaries were defined by the modified histones. To this end, we intersected the AS-SNP list with annotated elements from chromHMM and tfNet (see Methods). Across the different cell lines, we identified 17,293 unique AS-SNPs, located within annotated elements which were selected for further filtering Fig. . The majThe number of AS-SNPs identified for each cell line was highly correlated to the number of raw ChIP-seq reads aligned to the personal genomes, since better coverage increases the chances to detect allele specific signals  with GWAS top hits. Genetic control of gene activity has been analyzed in lymphoblastoid cell lines and we used a collection of eQTL SNPs (eSNPs) to investigate our AS-SNPs following the same approach as for GWAS SNPs.We wanted to identify AS-SNPs that are likely candidates to explain associations to disease in GWAS and to expression in studies of eQTLs (see Genomic Features in Methods). Therefore, the selected AS-SNPs were intersected with the SNPs with the strongest association (GWAS top hit) to selected B cell related traits from the GWAS catalog and with SNPs in high LD  were identified as allele specific with 603 AS-SNPs (66 in HLA region) reported in high LD. As shown in Table We also intended to identify those that are located in the same regulatory elements as an AS-SNP that is a candidate to a GWAS or eQTL signal. We therefore intersected rare AS-SNPs with windows (±300 bp) centered at common AS-SNPs associated to traits or eQTLs. The results are reported in Supplementary Table From the genetic point of view, immune system diseases represent the largest group of disease associated SNPs reported in the GWAS catalog, however these variants are in most cases the result of a statistical association and not the real functional SNPs driving the association. In this study, we identified AS-SNPs in high LD with GWAS SNPs associated with 20 unique B cell related traits/diseases Table . In mostNot surprisingly, many autoimmune disease related AS-SNPs were located in the human leukocyte antigen (HLA) region on chromosome 6. This highly polymorphic region, while essential for the adaptive immune system, has historically been challenging to study at a genomic regulatory level. Many GWAS and eQTL variants have been reported in the HLA region but the molecular mechanisms behind the associations remain elusive.DQA1 and DQB1 which have been reported as coding for HLA epitopes most strongly associated with susceptibility for T1D31. Thus, our data suggest that expression varies between HLA alleles and that this may contribute to the risk of T1D and other immune diseases.One example is the GWAS SNP rs9272346 reported in the GWAS catalog as associated to T1D however, this variant is not located in a regulatory element rich in TFBS and it is located in the highly variable HLA region , SLE (4 loci), RA (3 loci), amyotrophic lateral sclerosis (4 loci), vitiligo (3 loci) and celiac disease (4 loci).Outside the HLA region, in most cases, the loci defined by AS-SNPs were in close proximity with genes relevant for the disease, making them the primary targets for experimental validation of the regulatory mechanisms.RMI2 gene, important for gene stability and linked to celiac disorder32, two AS-SNPs (rs17035374 and rs12468278) located in two regulatory elements associated to the PLEK gene33, the AS-SNP rs6776027 in an intron of the FRMD4B gene, a reported celiac locus34 and the AS-SNP rs10511390, located in an intron of the ARGAP31 gene involved in intestinal barrier function and linked to celiac disorder35.For instance, for celiac disease, we identified four loci at non HLA-genes including the AS-SNP rs2216763 located in an intron of the C5orf30, a reported negative regulator of tissue damage in RA36. Two other AS-SNPs on chromosome 6, rs28362859 and rs2233434 were located in the third intron and first exon of the NFKBIE gene, encoding for the nuclear factor-κB (NF-κB) inhibitor epsilon. The NF-κB pathway is well documented as active in acute inflammatory responses and in chronic inflammatory diseases like RA37. Finally, a locus on chromosome 9 was flagged by two more AS-SNPs, rs3761846 and rs3761847 located in an intron of TRAF1, coding for the tumor necrosis factor-receptor associated factor 1 and highlighting how adding more pieces of information into the regulation of the TNF pathway is crucial to better understanding this complex disease.Another example are three loci associated to RA. Two AS-SNPs, rs28157 and rs28158, were identified in the same regulatory element on chromosome 5. They are located in an intron of We also identified 61 AS-SNPs associated to “multiple traits” e.g. the AS-SNP rs11557466 identified in GM18526 and reported in Table In this study we identified AS-SNPs defined by ChIP-seq reads from CTCF and SA.1, two architectural protein involved in the topological organization of the genome. SA.1 is part of the cohesin complex, a ring-shaped protein complex which is central in the 3D organization of the chromatin. DNA loops are “extruded” through the cohesin ring to form TADs and the TADs boundaries are marked by the transcription factor CTCF, whose binding motifs oriented in divergent directions acts as “anchor” points for cohesin ring. The sites at which CTCF binds are called interaction domains and the sequences in between form a loop (TAD loop). Most interactions between promoters and enhancers happen within these loops Fig. . We usedHiCap is a modification of HiC in which the ligated DNA molecules are hybridized to an array of candidate regulatory elements such as promoters of all genes and/or selected enhancers. We utilized in-house HiCap data in GM12878 generated with probes placed at the promoter regions of genes to identify all the distal elements interacting with a promoter and in particular regulatory elements with AS-SNPs. We found 173 unique AS-SNPs associated to gene expression and 60 to B cell related traits in distal regulatory elements that interact with defined promoters. This points to specific genes likely regulated differently between alleles. We also designed HiCap probes located in regulatory elements with AS-SNPs to detect interactions with promoters or other enhancers. In this more limited data set we found six additional AS-SNPs associated to gene expression and two to B cell related traits in distal elements that form 3D interactions with promoters.ZBTB38. rs724016 is located in a multi loop TAD defined by HiC data. The TAD spans ~0.3 Mb and contains two different genes (RASA2 and ZBTB38). HiCap data show that the probe harboring rs724016 interacts with the promoter of ZBTB38 confirming the association observed in the eQTL study. This highlights the screening power of long-range interaction data in defining the target(s) of regulatory elements. Motif analysis (see below) showed also how rs724016 altered binding sites for two TFs, BATF and NDF2, suggesting a putative molecular mechanism for the regulatory activity. In another 94 cases, HiCap data detected an interaction with a target gene different from the one detected in eQTL studies suggesting how HiCap can help establish the most likely target gene. An example is the AS-SNP rs1257573  and suggest TFs with differential binding.We also used the data to prioritize gene targets for AS-SNP associated to diseases via GWAS. An example is two AS-SNPs, rs11811536 and rs12032504 . Putatively altered TF-BSs were predicted for 98 (41.3%) and 325 (45.5%) of the AS-SNPs associated to GWAS or eQTLs respectively . It is crucial to keep in mind that the architectural regulation of gene expression might be more complex than the association reported by eQTL studies and that integrating long-range interactions data should be considered as a complementary approach when using eQTL data to inform GWAS signals on possible target genes.In order to identify the TF(s) whose binding could be altered by the presence of AS-SNPs we performed motif analyses using PMMs that take into account intra-motif dependencies. The idea is to evaluate whether BSs for specific TFs are overrepresented at regulatory elements harbouring AS-SNPs. Applying this kind of analysis to subsets of AS-SNPs associated to specific diseases via LD with GWAS SNPs could possibly define sets of TFs relevant for the disease pathology.These predictions, however, depend on (i) the choice of the significance-based threshold affecting the number of predicted BSs, (ii) the size of the genomic regions around SNPs considered, and (iii) the selection of TFs for which PMMs are built. Moreover, since the number available ChIP-seq data for TFs required for learning motif models is still rather limited, current predictions likely underestimate the number of BSs affected by AS-SNPs.In conclusion, we presented a systems strategy to find functional gene regulatory variants, the TFs that bind differentially between alleles, and novel strategies to detect the regulated genes. The collection of AS-SNPs presented here offers a set of promising candidate regulatory variants supported by several layers of evidence to prioritize experimental validations aimed at improving the knowledge of the molecular mechanisms of many immune-mediated diseases.30. The data sets were downloaded in short read archive format (.sra) from Gene Expression Omnibus (accession no. GSE50893). The reads quality was assessed using Phred64/33 scores with a quality cutoff requirement of 20.We used publicly available ChIP-seq data generated in 7 lymphoblastoids cell lines  for three histone modifications: H3K27ac, H3K4me1 and H4K4me3 and two genome architectural proteins: CTCF and SA.111 that takes a list of phased variants and a reference genome as inputs. We utilized the 1000 Genomes phase 3 phased genotypes for variants called in the selected 7 cell lines.  and the Genome Reference Consortium Human genome build 37 (GRCh37) as the backbone reference to build two in silico personal genomes for each cell line. ChIP-seq reads aligning to the reference and alternative genomes are referred to as G1 and G2 in Supplementary Tables We use the diploid genome reconstruction module from the ALEA toolbox6 which was completely rewritten in Perl and is available on http://bioinf.icm.uu.se/repositories.php. The pipeline is composed of 5 modules, each handling one or more aspects of the process. The division into separate modules is aimed at lower the computational cost. All settings and controls are handled via a single configuration across modules (see Extended Methods in Supplementary Information for more details).The AS-SNPs discovery was adapted from our established pipeline6 from the NHGRI GWAS catalog43. A total of 1545 unique GWAS SNPs were retrieved plus 26300 SNPs in high LD (r2 > 0.8) with them.GWAS SNPs associated to selected B cell related traits Table 6 from th44. A total of 5565 unique eQTL SNPs were retrieved from the GEUVADIS databse  plus 66935 SNPs in high LD (r2 > 0.8) with them.Collections of eQTL SNPs associated to gene expression in lymphoblastoid cell lines1000 Genomes SNP collection 45, centromeric and telomeric regions and GM12878 CNVsList of signal artifact blacklisted ENCODE regions46RegulomeDB47/tfNet48. Annotated regions were collected from tfNet where chromatin states were condensed into 7 categories: enhancer, heterochromatin, insulator, mixed, promoter, repressed and transcribed. AS-SNPs were selected when located into enhancers, promoters, mixed or insulators regions.ChromHMMDNase I hypersensitive sites (DHSs) in GM12878 were downloaded from the Gene Expression Omnibus (GEO): study accession code is GSM816665, ENCODE file accession code: ENCFF001UVC.AS-SNP collections were intersected and filtered with several publicly available databases:Previously, candidate target genes were selected by proximity with the GWAS SNPs but recent advances in studies of long-range 3D chromosomal interactions now offer new tools to detect direct evidence of interaction between promoters and their distal regulatory elements. Such experiments are done in cells or tissues that were fixed and chromatin fragmented using a restriction enzyme and the DNA ends ligated under extremely diluted conditions to create intra- rather than inter-molecule ligations. The DNA is then processed for paired-end sequencing and the reads were aligned to establish which ones were derived from promoter and enhancer sequences, thereby establishing long-range interactions.49 to remove invalid and duplicate pairs  in at least two of the replicates were retained. In total 130,915 interacting anchor loci were obtained. Distal interactions were defined as regions that interacted with the designed probes (promoters) or other distal genomic regions. Therefore, the final set contained regions that directly interacted with the designed probes (direct interactions) and regions that indirectly interacted with the designed probes (indirect interactions) through one or more distal regions (known as “hops”). AS-SNPs that overlapped the interacting regions  were further analysed.et al.51. For each TAD, contacting domains were defined as the two interacting anchor points. Loop domains were defined from the end point of the first contact domain to the start point of the second contacting domain. Genes that were located within each loop domain were assigned to the corresponding loop. Loops that contained the same sets of genes were unified to remove duplicates. Additionally, genes that overlapped the contacting domains were assigned as the anchor genes for the overlapping contacting domain.Additionally, we obtained Topologically Associated Domains (TADs) for GM12878 from Rao 52, we joined all training and test data sets (separately for each TF) from the HOCOMOCO database17  and learned PMMs by the de novo module of the InMoDe software52 using default parameters, yielding a total of 404 PMM models for 392 TFs. Six TFs  did not yield a single prediction on the input files for the given p-value threshold and have not been included in the analyses.For building a large collection of PMMset al.)48 aimed at identifying putative regulatory regions based on ENCODE data. We observed that when the distance threshold between consecutive (ChIP- or DNase-seq) peak summits was set to 300 bp it was greater than the distance between 87% of the input TFBS data essentially suggesting that the majority of the regulatory regions identified were ~300 bp in length. For this we used the Bioconductor packages GenomicRanges53 and BSgenome.Hsapiens.UCSC.hg19  and output for each AS-SNP two 601 bp sequences, one centered at the reference allele and the second at the alternative allele of the AS-SNP.We extracted the genomic sequences surrounding bi-allelic AS-SNPs in a window of 300 bp up- and downstream from the polymorphic site. The choice of 300 bp windows was driven by our previous work  di-nucleotide shuffling of all input sequences, (ii) computing all scores on the shuffled sequences, (iii) fitting a Gaussian density to the distribution of scores, and iv) determining the p-value of a given motif score from this Gaussian distribution. Predictions were filtered by a p-value of at most 1EFor each input sequence with a prediction above the threshold for at least one of the variants, we collected the motif scores and associated p-values for both variants. For the following analyses, we only considered binding site predictions that are affected by a specific SNP, i.e., that overlap a SNP and, hence, show a difference in motif score and p-value.Supplementary InformationTable S1Table S2Table S3Table S4Table S5Table S6Table S7Table S8


Acquired Immunodeficiency Syndrome (AIDS) has been devastating for millions of people around the world. Inhibition of the human immunodeficiency virus (HIV) protease is among the most important approaches for the therapeutic intervention in HIV infection. Since the discovery of the HIV-1 protease, this enzyme has been considered as a key target for the inhibition of viral replication. A large body of research has been done to develop an effective HIV-1 protease inhibitor. There are to date 10 HIV-1 protease inhibitor drugs approved by the Food and Drug Administration (FDA) that have improved the survival and quality of life of HIV infected people. These drugs are prescribed in combination with the reverse transcriptase inhibitors, which is referred to as highly active antiretroviral therapy (HAART). The HIV-1 protease inhibitors play a vital role in HAART. The applications of click chemistry are dispersing in the field of drug discovery. Recently, click chemistry has captured a lot of attention and has become a powerful tool for the synthesis of medicinal skeletons in the discovery of anti-HIV drugs. Click reaction is a well-known method for making carbon−heteroatom−carbon bonds. Click reactions are popular because they are wide in scope, of high yielding, quick to perform, and easy to purify. In this review, we outlined current approaches towards the development of HIV-1 protease inhibitors employing click chemistry. The first case of AIDS was reported in the United States in June 1981. AIDS became an epidemic that affected millions of people around the world . Since tHIV is a retrovirus that if left untreated can lead to AIDS . HIV sprAntiretroviral therapies (ART) are medications that do not kill the HIV virus but can prevent the growth of the virus. When the growth of the HIV virus is slowed down, the HIV disease progression also slows down. There are six main types of antiretroviral drugs that currently exist based on the stage in the viral life cycle where they are targeted: (i) nucleoside reverse transcriptase inhibitors (NRTIs) and nucleotide reverse transcriptase inhibitors (NtRTIs), that work by blocking the reverse transcriptase enzyme so that HIV cannot make new virus copies of itself; (ii) non-nucleoside reverse transcriptase inhibitors (NNRTIs), which work by blocking the enzyme reverse transcriptase and prevent reverse transcription, thus stopping HIV replication; (iii) protease inhibitors (PIs), that work by blocking the activity of protease enzymes. HIV uses enzyme protease to break up large polyproteins into smaller pieces, which are required for the assembly of new viral particles .The HIV can still replicate but the resulting virions are immature and cannot infect new cells ; (iv) fuCombination of the antiretroviral drugs is referred to as HAART, considered as the most effective treatment model for AIDS. Protease inhibitors play a very important role in HAART . After tDrugs that are designed to attack HIV-1 protease are among the stellar breakthroughs of modern medicine. HIV protease's protein structure was first reported in 1989 and later has been deeply studied and investigated using X-ray crystallography. The studies of X-ray crystallography revealed the structure of HIV-1 protease exists as a homodimer . A waterThe advancement of crystallographic techniques improved the understanding of HIV-1 protease structure and made it the most well-studied enzyme known to medicine. The HIV-1 protease is a retroviral aspartyl protease that is essential for the life cycle of HIV, the retrovirus that causes AIDS . HIV proRecently, click chemistry has emerged as a powerful tool in drug discovery to synthesize compounds containing carbon-heteroatom-carbon bonds , 19. CliThe click reactions are popular because click chemistry employs chemical reactions that are wide in scope and of high yielding and produce very little byproduct that can be removed easily by chromatographic techniques. These reactions are quick and easy to perform and are stereospecific. Carbon-carbon double bond or carbon-carbon triple bond provide energy for click connection and therefore serve as a starting material for click reactions. Click chemistry is the powerful reaction for making carbon−heteroatom−carbon bonds  in an aqueous environment , 27. In 1) -) which makes it difficult for the protease to cleave the linkage. HIV protease inhibitors fit the active site of the HIV aspartic protease given the aspartyl protease's mode of action. The most promising transition state mimic was hydroxyethylamine, which led to the discovery of the first protease inhibitor, Saquinavir (1) .2), a peptidomimetic HIV protease inhibitor, was designed to fit the C2-symmetry in the binding site of the protease, while Nelfinavir (3) was the first protease inhibitor that was not peptidomimetic. It was designed as a nonpeptidic inhibitor; an iterative protein cocrystal structure of the inhibitors was replaced by nonpeptidic substituents . Sy. Sy et a et al. in 2013 reported the synthesis of another series of N-benzylated amido analog of coumarin-AZT conjugates (35a-e) with a similar chemical sequence ( N-benzylated amido group was introduced by treatment of the intermediates (29a-e) with benzylamine followed by treatment with chloroacetyl chloride. Nucleophilic substitution reaction of propargylamine with chloroacetamide (33a-e) afforded alkynylated products (34a-e), which upon subjection to Cu (I), catalyzed click chemistry with AZT, afforded coumarin-AZT (31) conjugates (35a-e).The ethylene dipeptide HIV-1 PR inhibitors usually contain benzyl groups. Therefore, Olomolasequence  37]. Th. Th et a N-benzylated cycloaddition products (35a-e) and a series of non-benzylated analogs (32a-e) as a twofold action of HIV-1 PR/RT inhibitor.Saturation transfer difference (STD) NMR, enzyme-inhibition, and computer modeling techniques have been employed to evaluate the potential of theThis review focuses on the application of click chemistry in the development of HIV-1 protease inhibitors. The applications of click chemistry are spreading, ranging from discovery through combinatorial chemistry to the field of proteomics employing the bioconjugation reactions. Click chemistry played a vital role in the antiviral drug discovery, and it can be anticipated that in the future it will make further contributions in anti-HIV drug discovery. Scientists around the world have been trying to develop the medicinal drugs to combat HIV. HIV medicines will help people to live longer, healthier lives and also reduce the risk of transmission and control the spread of disease. The HIV protease inhibitors continue to play a very important role in HAART. There are some side effects associated with the antiretroviral drugs, depending on the individual body type and the level of HIV infection. Therefore, drug therapies that are more efficient have fewer side effects and drug-related toxicities are required. This critical review summarizes the progress made so far in the discovery of HIV-1 protease inhibitors employing innovative approaches. The click chemistry-based approach has become a popular protocol to synthesize HIV-1 protease inhibitors. A lot of research is still needed in the area of antiretroviral drug development to discover new antiretroviral molecules with improved potency that will reduce side effects and provide a new mechanism of actions.


The data presented in this article is supplemental data related to the research article entitled “Automated external defibrillator accessibility is crucial for bystander defibrillation and survival: a registry-based study” . We present detailed data concerning: 1) the type of location for deployed and registered automated external defibrillators (AEDs) in the nationwide Danish AED Network; 2) the number of registered AEDs in the nationwide Danish AED Network, and changes in AED registration (according to year and type of AED location); 3) the number of AEDs being withdrawn from the AED network between the years 2007–2016. We also report data on baseline cardiac arrest-related characteristics of out-of-hospital cardiac arrests (OHCAs) that occurred in Copenhagen, Denmark, between 2008 and 2016. Cardiac arrest-related characteristics are further described according to AED accessibility (accessible vs. inaccessible AED at the time of OHCA) for OHCAs covered by an AED (AED ≤200 m route distance of an OHCA). Finally, we report data on distance to the nearest accessible AED for bystander defibrillated OHCAs covered by an AED ≤200 m route distance where the AED was inaccessible at the time of OHCA. Cardiac arrest-related characteristics were investigated according to whether the nearest AED ≤200 m route distance of the OHCA was accessible or not at the time of OHCA. Categorical variables were compared with the chi-square test, and continuous variables with the Kruskal-Wallis test. A 2-sided p-value <0.05 was considered significant. Analyses were performed using SAS .


They were then instructed to utilize similar ER strategies in an emotion reactivity task during which they viewed high arousing negative pictorial stimuli while their heart rate (HR), heart rate variability (high frequency power – HF-HRV) and subjective affective states were measured. First we found that the East Asian group reported higher use of interpersonal ER strategies of social modeling and perspective taking in daily life. During the experimental interpersonal prime exposure, the East Asian group showed elevated HF-HRV (relative to baseline) compared to the Western European group, indicating more adaptive ER, but this pattern was not sustained during the reactivity or recovery phases. Instead, the East Asian group demonstrated increased HF-HRV and decreased HR across both prime conditions. The East Asian group also showed greater decreases in positive affect across the course of the experiment. Furthermore, individual differences in social modeling and individualistic self-construal moderated the effect of the ER prime in the East Asian group at trend levels, and main effects for perspective taking and reappraisal were observed in the Western European group. The findings support the notion that engaging in interpersonal ER strategies may be more beneficial for East Asian groups when immediately exposed to a stressful situation, as these strategies are congruent with cultural context and preferences, but our priming methodology may have limited the longer-term benefits.Cultural differences exist in the use of emotion regulation (ER) strategies, but the focus to date has been on intrapersonal ER strategies such as cognitive reappraisal. An emerging literature highlights the importance of interpersonal ER, which utilizes social cues to facilitate the regulation of emotional states. In cultures that place high value on social interconnectedness as integral to their collectivistic self-construal, including East Asian cultures, interpersonal ER strategies may be particularly effective in reducing negative affect but this has not been previously tested. In this study, two groups comprising East Asian ( Emotion regulation (ER) functions to shape how we respond to, express and manage our emotional responses to external and internal events. A multi-faceted process, ER works by increasing, decreasing or maintaining the intensity or experience of an affective state in order to navigate our social environment, cope with internal stress or external threats . TheoretResearch suggests that social context is critical to informing the effectiveness of ER strategies , and thiResearch has shown that individuals engage in and benefit from employing intrapersonal ER strategies that are consistent with cultural goals and self-concept . As suchCulture may also influence the use of interpersonal  ER practices, but thus far, it is unknown whether there are cultural differences in the implementation or benefit derived from engaging in interpersonal ER strategies. More broadly, there has been relatively less attention paid to the interpersonal aspects of ER compared to intrapersonal strategies. The emerging notion of interpersonal ER seeks to incorporate the social factors that influence ER processes . InterpeWhile there has been some theoretical development of these concepts , alongsiAn outstanding empirical question is whether culture also affects how interpersonal ER strategies are engaged. Given a key mechanism differentiating cultural groups in terms of emotional expression, perception and social function is self-construal , which dThis study aims to examine this question by comparing engagement in interpersonal ER practices in groups of Western European (WE) and EA participants. First, we were interested in whether groups differed in self-report measures of habitual use of intrapersonal and interpersonal ER strategies. In our first hypothesis, we predicted that EA participants would report engaging in interpersonal strategies such as perspective taking and social modeling more frequently than the WE cohort, due to stronger levels of collectivistic self-construal and interdependent social orientation.Second, we were interested in whether groups differed on the impact of engaging in interpersonal ER strategies to manage affective and physiological reactions to negative cues. Effective ER can be measured via self-report , or physiologically. Heart rate variability  is a well-evidenced physiological indicator of adaptive self- and ER . AccordiIn the current study, participants in each cultural group were randomly assigned to be primed with a stress-induction scenario where the narrator engaged in either interpersonal or intrapersonal ER strategies. Following this, participants viewed high-arousing negative scenes in an emotion reactivity paradigm, while their HR was recorded and subjective ratings of emotional state were measured. In our second hypotheses, we predicted that the EA group would show greater benefit from implementing interpersonal ER strategies during the prime and while viewing negative cues compared to the WE group and relative to intrapersonal strategies. By contrast, the WE group would demonstrate greater benefit from implementing intrapersonal ER strategies during the task compared to the EA group and interpersonal strategies. This benefit would be reflected in (a) lowered subjective negative affect and (b) reduced HR and elevated HRV (indicating adaptive ER). In our third set of hypotheses, we predicted that individual differences in habitual use of interpersonal and interpersonal ER, and self-reported levels of individualistic or collectivistic self-construal, would moderate the degree that engagement in either interpersonal or intrapersonal ER strategies influenced negative affect or HR outcomes in each cultural group. For instance, habitual use of interpersonal ER strategies like perspective taking or social modeling might be expected to enhance the effect of the interpersonal prime.n = 6), participants who reported bicultural heritage (n = 4), and having South Asian (n = 1) or Southern/Eastern European (n = 3) ancestry). Participants were also subsequently excluded if they did not meet the inclusion criteria on a number of key factors that may affect HR and emotional responses which were measured during the study (see below): regular recreational or medicinal drug-use (n = 2); current smoker (n = 6); or scores on any of the subscales of the Depression Anxiety Stress Scale indicating extreme symptom severity  ancestry, or EA  ancestry. Further inclusion criteria included identity with a single cultural background  and for the EA group, living in Australia for less than 10 years. Fourteen participants had to be subsequently excluded due to incorrectly responding to pre-screening questions and not meeting cultural group inclusion criteria . There were 48 participants in the EA group  and 38 participants in the WE group . Even if excluded, participants received either course credit or AUD $15 in return for taking part in the study.Thus, a final sample of 86 participants completed the study  Human Research Ethics Advisory Panel C: Psychology. All participants gave informed consent in accordance with the Declaration of Helsinki.Participants were administered a number of self-report measures to index various aspects of culture and ER capacity.Interpersonal Emotion Regulation Questionnaire (IERQ) (‘I like being around others when I’m excited to share my joy’), perspective taking , soothing , and social modeling . Each item was rated on a 5-point Likert scale  and responses were summed within the four subscales. The original psychometric validation of the IERQ was conducted on a mixed cultural group cohort, including respondents with Asian ethnicity (The e (IERQ)  measuresthnicity . The intEmotion Regulation Questionnaire (ERQ) is a 10-item measure of the use of intrapersonal ER strategies of reappraisal and emotional suppression to manage emotions in day-to-day life . Adaptation of the ERQ to Asian youth populations indicate support for the original factor structure of the instrument , with items summed within subscales. The internal consistency for this sample was sound for both subscales; collectivistic/interdependent (Cronbach α = 0.72) and individualistic/independent (α = 0.71). The SCS has been widely used and validated in EA populations . The VVIQ has been used in studies with EA participants . Researchers have demonstrated cross-cultural construct validity of the DASS-21 in Asian groups , and internal consistency was satisfactory for the interpersonal (Cronbach α = 0.76) and the intrapersonal subscales (Cronbach α = 0.61).A post-manipulation measure consisting of eight items was used to index the impact of the prime. These questions described different interpersonal  and intrapersonal  ER strategies. Participants rated how frequently they engaged in each strategy during the emotion reactivity phase on a 5-point Likert scale (1 = “They (emergency workers) do not look too troubled about the situation which helps ease my worry” and “I notice a girl from one of my lectures standing in the crowd looking at the cars. She looks in my direction and flashes me a kind smile. This helps me to feel calmer”], perspective-taking and soothing . In the intrapersonal ER prime, various intrapersonal ER strategies were described including reappraisal  and distraction . Each prime was pre-recorded using the same narrator and played to the participant via headphones, and were 2 min in duration.Scenarios were developed to prime the implementation of either interpersonal or intrapersonal ER strategies when encountering a stressful experience. Each scenario featured the same first-person narrator approaching the scene of a motor vehicle accident on foot, with the narrator describing what they saw, how they felt and the ER strategies they engaged in. The two prime scenarios were matched on: their length, level of detail, the number of times ER strategies were used, and the ultimate outcome of applying the ER strategies . In the interpersonal ER prime, various interpersonal ER strategies were described that included social modeling  were then screened for outliers (defined as more than three standard deviations from the sample mean). Outliers were then replaced by either the maximum (three standard deviations above the mean) or minimum integer . This method is commonly used in psychophysiological studies to prevent outliers from unduly influencing statistical outcomes but also to retain the overall distribution in the data . Next, dDifference scores comparative to baseline for the three active phases were also calculated for positive and negative affect, similar to the HR/HRV data. Levene’s test of equality of error variance was statistically significant for positive affect during the prime phase. Therefore, the data was transformed through replacing outliers according to a strict protocol (1.5 standard deviations below or above the mean), with a total of 6.9% of the positive prime difference scores being replaced.t-tests were carried out to verify that the cultural groups differed on the subscales of the IERQ, ERQ, and SCS to reflect previous theoretical and empirical findings. Then cultural group (WE vs. EA) and manipulation group  differences on self-report measures of interpersonal and intrapersonal ER, self-construal, demographics and habits  that impact HR were examined using one-way ANOVAs comparing the four experimental groups for continuous data  and chi-square tests for categorical data (p < 0.05). For continuous measures, post hoc contrasts tested whether significant differences detected were between cultural or ER prime groups (Bonferroni-corrected).Data was analyzed using SPSS (v. 24). Firstly, independent p < 0.05). Age, alcohol and anxiety scores were included as covariates as these three factors were found to be significantly different between groups, and are known to effect HR and HRV  MANCOVA was conducted to examine between and within-group effects on HR, HRV and self-reported affect difference scores  as a function of culture and ER prime groups  × 2  ANCOVAs were subsequently conducted to examine between-group effects at each phase  separately, followed by pairwise comparisons to determine the direction of the significant effects .p-value less than 0.1; in the final models, significant predictors were determined at p < 0.05. Simple slopes analyses were carried out as post hoc tests to further examine the meaning of a significant interaction term .Finally, a series of regression analyses were conducted to determine the relative contribution of individual differences in self-construal  and habitual self-reported use of interpersonal  and intrapersonal  ER strategies on HF-HRV during each phase of the study. As such, hierarchical moderated regression analyses were conducted for the two cultural groups separately. Predictors were mean centered to correct for multicollinearity. Two-way interaction terms were included in the model to examine the interaction between prime group and each predictor. Initial hierarchical moderated regression models consisted of three steps: (1) prime group; (2) independent mean centered predictors; (3) interaction terms between prime group and each predictor value. From these initial regression models, significant predictors were identified and included in final regression models. A significant predictor in the initial model was defined as any predictor with a SD = 2.78). Chi-square tests indicated that were no significant group differences between caffeine  or energy drink  consumption. Groups significantly differed in terms of age, with post hoc contrasts indicating that those randomized to the interpersonal prime condition were older than those in the intrapersonal prime condition . Groups were similar on the depression and stress subscales of the DASS, but differed in terms of anxiety, with the EA group reporting higher anxiety scores than the WE group . Groups also differed in terms of alcohol consumption, with post hoc contrasts demonstrating that the WE group reported higher levels of alcohol use than the EA group . As such, subsequent ANOVA tests controlled for group differences in age, anxiety and alcohol use.t-tests revealed that the EA group reported higher levels of collectivistic self-construal compared to the WE group , but no group difference in individualistic self-construal was observed .In regards to self-construal, the one-way ANOVA did not reveal significant differences, but we did not expect there to be differences between prime group. Considering just cultural group, F = 4.92, p = 0.003]. Post hoc contrasts indicated a significant main effect of cultural group: EA participants reported using interpersonal strategies more frequently during the experiment than WE participants, regardless of their manipulation group . There was also a significant main effect of manipulation condition. Participants in the interpersonal condition reported using interpersonal strategies more than participants in the intrapersonal group , suggesting the prime narration activated interpersonal ER strategies. However, a similar effect was not observed for those in the intrapersonal group (suppression – t(82) = −1.06, p = 0.29; reappraisal/distraction – t(82) = 0.46, p = 0.65).Participants answered post-prime manipulation questions to indicate whether the experimental prime had an influence on the ER strategies used during the experiment. A one-way ANOVA conducted across the four groups indicated that there was a difference in the self-reported use of interpersonal ER strategies between groups  or suppression  between groups. Notably, there was a marginally significant effect for the suppression condition, indicating a trend toward the EA group reporting greater use of suppression relative to the WE group.There were no significant differences between the four groups in habitual use of intrapersonal ER strategies , and culenhancing positive affect, social modeling, and perspective taking (p < 0.05). Post hoc contrast tests showed EA participants had higher perspective taking  and social modeling scores  than WE participants, averaged across manipulation groups, suggesting that EA group more commonly engaged in these two core interpersonal ER strategies in daily life. Post hoc contrast tests also revealed that WE participants had higher enhancing positive affect scores than EA participants .The ANOVA revealed that there were statistically significant group differences on three of the IERQ subscales: To determine whether groups differentially benefited from engaging in interpersonal vs intrapersonal ER following the prime, a 2 × 2 × (3) MANCOVA was conducted. The between-subjects factors were cultural group (WE and EA), and prime group , and the within-subjects factor was experimental phase . Covariates were age, alcohol and anxiety scores. The dependent variables were self-reported positive and negative affect change (relative to baseline).F = 6.07, p = 0.003]. Post hoc MANCOVAs conducted within each phase found that while cultural group differences were not detected during the prime phase , significant main effects of culture on positive affect change following the emotion reactivity  and recovery phases  = 10.25, p=.002) were evident (Bonferroni-corrected). This effect was due to stronger decreases in positive affect being reported in the EA group compared to the WE group during emotion reactivity and recovery relative to baseline. No significant between-group or interaction effects were observed for the negative affect change scores.A significant cultural group by phase interaction was detected for positive affect  and HR  were found, indicating that the EA group demonstrated elevated HR-HRV and reduced HR relative to the WE group across all phases of the study. There was also a significant main effect of prime group on HR , such that the intrapersonal prime group showed greater decreases in HR relative to baseline across all phases of the study compared to the interpersonal prime group.No significant three-way or two-way interaction effects for either measure were observed. Significant between-subjects main effects of cultural group on HF-HRV . Follow-up pairwise comparisons indicated that EA participants showed elevated HF-HRV (absolute units) compared to WE participants in the interpersonal ER condition (p = 0.018). A significant between-subjects cultural group main effect at the prime phase on HR , revealed greater decreases in HR relative to baseline in the intrapersonal ER condition across groups; see A significant between-subjects cultural and prime group interaction for HF-HRV during the prime phase was observed  and HF-HRV ; see Significant cultural group main effects during the emotion reactivity phase were observed for HR  and HF-HRV  was detected. Again, EA participants showed reduced HR and elevated HF-HRV compared to WE participants, but no interaction effects with prime group were found  changes during the prime, emotional reactivity and recovery phases of the study. We focused on HF-HRV (absolute units) as the key dependent variable as this was where we observed a cultural by prime group interaction in the between-group ANCOVA analysis (prime phase only).F = 2.476, p = 0.039, Adj R2 = 0.16] and accounted for 26.6% of the variance. The prime group was a significant predictor, indicating that EA in the interpersonal prime condition showed elevated HF-HRV compared to those in the intrapersonal condition.The final model was significant  and accounted for 24% of the variance of change in HF-HRV during emotion-reactivity in the EA group. Individualistic self-construal in interaction with prime group emerged as the strongest predictor. Simple slopes analysis showed that levels of individualism did not affect HF-HRV in the interpersonal prime condition , however, higher individualism predicted greater change in HF-HRV relative to baseline for those in the intrapersonal prime group ; see The final model was significant at the third step  and accounted for 10.9% of the variance in HF-HRV change during the prime phase for the WE group. Perspective taking was the only significant predictor as a main effect, suggesting that the lower the self-reported perspective-taking, the greater the change in HF-HRV during priming across both conditions.The final model was significant  and accounted for 22.1% of the variance in HF-HRV change during recovery. Higher levels of trait reappraisal and lower levels of perspective taking were associated with elevated HF-HRV relative to baseline during the recovery phase in the WE group.The final model was significant [This study examined whether culture moderated the benefit of engaging interpersonal ER strategies during a prime and emotion reactivity task. The findings support the notion that an EA cultural group with stronger collectivistic self-construal, reported greater habitual engagement in social modelling and perspective taking interpersonal ER strategies compared to a WE group. Moreover, priming interpersonal ER strategies to manage emotional reactions to negative stimuli increased HF-HRV – an indicator of parasympathetic innervation of the heart and of stronger ER – in the EA compared to the WE group during exposure to the prime itself, but not during the subsequent emotion reactivity or recovery phases. This suggests that the EA group benefited more from interpersonal priming relative to the WE group in terms of physiological responding, but that this did not extend beyond the immediate prime exposure phase. Furthermore, the EA group demonstrated generally higher HF-HRV and lowered HR during reactivity and recovery phases compared to the WE group, indicating the EA group was better at implementing both interpersonal and intrapersonal ER strategies to moderate physiological responses to negative cues. The findings also support the role of individual differences in self-construal, interpersonal and intrapersonal ER styles in modulating HF-HRV responses across the phases of the experiment within each cultural group.implicit forms of social support  when managing stress reactions  and perspective taking  during both priming and recovery phases in the WE group. Previous studies have found the use of ER strategies associated with consideration of others  or placing high value on interpersonal harmony was negatively correlated with the wellbeing of WE individuals . SimilarThe findings of this study must be interpreted within the context of a number of limitations. We found that the prime condition did not modulate cardiovascular or affect indices beyond the prime exposure phase, suggesting that the priming method employed in this study may not be the most effective approach for studying the effect of engaging in interpersonal ER strategies to manage emotional reactions to negative cues. This approach was selected to maximize the study of implicit cultural differences in ER strategy engagement, due to previous research demonstrating the maladaptive effects of explicitly instructing ER strategies if these strategies are incongruent to trait ER style . Future To our knowledge, this is the first study to consider cultural differences in interpersonal ER. The findings support the notion that an EA cultural group reported stronger engagement in interpersonal ER strategies such as social modeling, and demonstrated cardiovascular indicators of more adaptive ER responses during an interpersonal ER prime, compared to a WE group. The findings highlight the potential for interpersonal ER strategies to enhance the management of emotional reactions particularly in collectivistic cultural groups, adding to the literature that the benefit of various ER strategies may be dependent on various contextual factors – including cultural congruence.The research was approved by the UNSW Human Research Ethics Advisory Panel C: Psychology. Participants provided written informed consent after reading the Participant Information and Consent Form, and had the freedom to withdraw from the study at any time. Once completed, participants were fully debriefed and received either course credit or AUD $15 reimbursement.BL conceptualized and designed the study, supervised data collection and analysis, and wrote the manuscript. EW conceptualized and designed the study, conducted data collection and analysis, and contributed to the manuscript based on her Honors in Psychology thesis.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.


Schwannoma is a rare source of false-positive 18F-fluorodeoxyglucose (18F-FDG) uptake in Positron-emission tomography (PET/CT), inducing potential errors in staging of several solid cancer, with implications for patient management. This clinical case reports the situation of a patient undergoing an 18F-FDG-PET/CT for initial staging of an ovarian adenocarcinoma. We found a high paramediastinal hypermetabolic mass suspicious of remote extension or secondary synchronous primitive tumor. The biopsy finally reveals a histopathology of Schwannoma, allowing the patient to be eligible for a surgical procedure of her ovarian adenocarcinoma by rejecting the hypothesis of malignancy. Even if they are considered as benign tumors, high FDG uptake can be seen in schwannomas, providing a risk of false-positive interpretation with consequences on tumor staging and also management of patient.For example Martinez-Esteve et al.  have repMoreover, Gorospe et al.  have pubSeveral other case reports show that schwannomas are usually associated with a high 18F-FDG uptake on PET-CT –6. But, Only few cases of schwannoma located in the mediastinum with an 18F-FDG uptake have already been reported in literature.A 75-year-old female patient with previous hystory of active smoking at 75 year-package associated with other cardiovascular risk factors  has been sent to our university hospital for a suspicion of strangulated umbilical hernia. An abdomen and pelvis CT scan was then performed and found a diffuse infiltration of mesenteric fat evoking a peritoneal carcinosis without primary tumor clearly identified.An exploring laparoscopy showed a visual aspect of inflammatory peritoneum with a thickened epiploon and non-tumorous ovaries. On the contrary, histopathological examinations (biopsy and cytology) suggested an immunohistochemical profile compatible with high-grade serous papillary carcinoma of ovarian or peritoneal origin. The therapeutic strategy included neo-adjuvant chemotherapy by CARBOPLATIN-PACLITAXEL and interval surgery after 3 cycles.Moreover, an 18F-fluorodeoxyglucose (18F–FDG) Positron-emission tomography (PET/CT) was performed not to ignore a supra-diaphragmatic remote extension of disease that would exclude surgery indication. In addition to multiple hypermetabolic known peritoneal carcinomatosis lesions Figure –4. Due tWhile awaiting the histological characterization of this mass, the patient finally benefited from 6 cycles of chemotherapy before surgery by laparotomy. Unfortunately due to carcinomatosis extended to the entire abdominal cavity with a peritoneal index at 19  a compleSchwannomas are the most common nerve sheath tumors  and are This tumor consists of an abnormal proliferation of Schwann cells developped from nerves. The most frequent locations of schwannomas are the brachial plexus or the large nerve trunks of the limbs . Deep forms in retroperitoneum or mediastinum exist and are often of large size . In the On imaging, schwannomas occur on the path of a nerve, are well-limited, often oval and unique. The metabolic characteristics of schwannomas on 18F-FDG PET-CT are not well-described in literature to differentiate benign or malignant forms. Ahmed et al. did not found an association between SUV and benign schwannoma or malignant tumor .n = 9). They found that the SUV max of the hypocellular tumors was significantly lower than the SUV max of the hypercellular tumors . They did not found an association between SUVmax and tumor size or Ki-67 index. They did not find why there is a high FDG accumulation in benign tumors such as schwannoma.Beaulieu et al.  comparedp = 0.007) and the presence of peri-tumor lymph nodes (p < 0.001).In a study of 22 histologically proven schwannomas, Miyake et al.  uptake  and alsoIt therefore seems difficult to conclude to a diagnosis of Schwannoma only based on SUV characteristics on PET-CT since their SUVmax seems to vary enormously. There is also several other causes of known false positives such as infectious and inflammatory processes.Several cases reporting masses to PET-CT with hypermetabolism in follows-up of tumoral pathologies have been described, and these hypermetabolisms are generally associated with malignant tumors. However as described earlier that is not always true. Schwannomas with an 18F-FDG uptake have been described but especially outside the mediastinum. To our knowledge only few publications report this situation in the mediastinum. This case confirms the necessity to be aware of a neurogenic tumor when the PET reader concludes to a hypermetabolic mass of the posterior mediastinum and especially if it can lead to a change in patient care.Schwannoma is a potential cause of 18F-FDG false positive uptake in PET-CT, inducing risk of worse staging or therapeutic assessment, with consequences on patient management. Currently there are no reliable argument to differentiate a benign Schwannoma from a malignant tumor only on 18F-FDG-PET-CT imaging. This case report also recalls the need to biopsy doubtful lesions fortuitly discovered on 18F-FDG-PET-CT in order to avoid a loss of chance and possible complications related to unnecessary treatment toxicities.All procedures performed in this study were in accordance with the ethical standards of the institutional research committee on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008. Ethical review and approval was not required in accordance with the national and institutional requirements. The patient provided written informed consent.PB provided details of the patient and provided initial draft of submission. RD provided details of the patient and helped draft the initial submission. P-YL and RA provided images, image analysis, and helped draft the initial submission. LO helped draft the initial submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The molecular mechanisms regulating the endothelial response to high doses of radiation are suggested to be initiated via translocation of endothelial asmase from the inner to the outer leaflet of the plasma membrane. Further, asmase catalyzes the hydrolysis of sphingomyelin to generate the lipid second messenger ceramide that leads to the transmembrane signaling of apoptosis 5]. S[5]. Sasm. S[5]. Spathways . The mecw-dose 1.-3 Gy fraw-dose 1.-3 Gy fraasmase activation causes vascular collapse followed by endothelial apoptosis to be mandatory for tumour growth inhibition. Contrarily pretreating endothelial cells with the ceramide antagonist sphingosine-1-phosphate (S1P), reverses the asmase/ceramide-induced cell death process [Several studies have now confirmed radiation-induced  process . Multipl process .et al. investigated the role of asmase-ceramide mechanotransduction by combining USMB with radiation (2 and 8 Gy) in vivo. Experiments were conducted in asmase wild-type, as well as chemically treated (SIP) and genetically modified endothelial apoptosis-resistant mice (asmase knockout) implanted with fibrosarcoma xenografts. Tumour response to treatment was monitored using quantitative 3D Doppler ultrasound and immunohistochemistry at 0, 3, 24, and 72 hours after treatment. Results from experiments with asmase wild-type mice, evaluated 24 hours after treatments demonstrated a decrease in tumour perfusion of up to 46% by three hours following radiation and USMB. The drop in tumour perfusion persisted for up to 72 hours and hence was directly linked to an increase in tumour cell death by 53% followed combination of radiation and USMB. In contrary, mice treated with S1P and asmase knockout mice revealed no statistically significant decrease in tumour perfusion and no concomitant changes in cell death [In a recent study published in the Journal of the National Cancer Institute, El Kaffas ll death .et al. provide preclinical evidence that the combination of radiation and USMB resulted in significant increases in ceramide generation followed with tumour growth delay in asmase wild-type mice. In contrast, S1P-treated or asmase knockout mice were reported with minimal ceramide staining and growth delay. It has been confirmed previously that the paradigm of a tumour vascular disruption leading to endothelial apoptosis is directly dependent on ceramide generation [et al. also reported the changes in the concentration of plasma asmase and different ceramide metabolites (ceramide synthase and ceramide kinase). Interestingly, the results revealed high levels of plasma asmase and ceramide synthase in asmase wild-type mice but not in groups treated with S1P followed radiation and USMB. In opposition, ceramide kinase, an enzyme that metabolizes ceramide to ceramide-1-phosphate was found to be elevated only in asmase knockout groups which correlates with the restoration of radioresistance in asmase knockout animals [El Kaffas neration . Additio animals . Further animals .et al. study demonstrate for the first time the mechano-acoustic activation of asmase/ceramide pathway followed radiation and USMB using genetic and chemical approaches. The paper also highlights some of the limitations of the study. The authors mention that the study lacks the usage of ideal techniques to distinguish between perfused and nonperfused vessels in histopathology. Incorporating perfusion assays as a gold standard histological assessment will help overcome this problem. Secondly, the study addresses the relatively poor resolution of small blood vessel imaging detected by 3D Doppler high-frequency ultrasound and how implementing contrast-enhanced ultrasound or optical imaging could provide with better resolution of blood vessels. Finally, to translate this study in clinical research requires proper planning to maximize the combined effect of radiation and USMB on tumours with minimum effects on healthy tissues. That can be achieved by the selective focussing of ultrasound to only stimulate microbubbles within a tumour target. Collectively, the findings of El Kaffas et al provide a basis for understanding the importance of asmase/ceramide in radiotherapy and mechanobiology.To summate, the results of El Kaffas


The insertion of the guidewires (GWs) into the pancreatic duct is technically difficult, and there is a risk of post-ERCP pancreatitis (PEP). The aim of this study was to evaluate the safety and efficacy of a small J-tipped guidewire for pancreatic duct endoscopic intervention. This single-site retrospective study was conducted to assess the procedural success rate and adverse events of endoscopic transpapillary interventions to the pancreatic duct in 114 cases using the small J-tipped GW and 180 cases using the angle-tipped GW. The procedural success rate was significantly higher in the small J-tipped GW group compared with that in the angle-tipped GW group . The procedural success-related factors were chronic pancreatitis , flexion angle of the pancreatic duct < 90° , and use of the small J-tipped GW . The rates of total post-ERCP pancreatitis were significantly lower in the small J-tipped GW group compared with that in the angle-tipped GW group . Multivariate analysis of pancreatitis risk factors indicated that only the use of the small J-tipped GW was a factor in decreasing the risk of developing pancreatitis . Small J-tipped GWs increase the success rate of the pancreatic duct endoscopic intervention as well as a reduced risk of developing postoperative pancreatitis. Endoscopic transpapillary interventions to the pancreatic duct (pancreatic duct endoscopic intervention), such as a detailed examination for pancreatic diseases and stenting for pancreatic duct stricture, became more common in recent years.Although pancreatic duct endoscopic intervention is usually performed with guidewire (GW) placed in the pancreatic duct, the insertion of a GW itself is difficult because the pancreatic duct is anatomically more complicated with advanced curves and strictures compared with the biliary duct . In addiPancreatic stent placement has been reported to be beneficial in preventing PEP because it prevents pancreatic outflow obstruction due to papillary edema –10. HoweRecently, J-tipped GW designed for improving luminal passage has been developed and reported to be beneficial in selective biliary cannulations , 13. AltThis study was conducted at the University of Yamanashi Hospital between January 2011 and September 2016. All patients underwent endoscopic transpapillary intervention with the GW insertion into the pancreatic duct for pancreatic disease or pancreatic GW technique . We used angle-tipped GWs from January 2011 to February 2014 and small J-tipped GWs from March 2014 to September 2016. The patients were divided into two groups according to treatment timing based on the type of GW used: the angle-tipped GW group (from January 2011 to February 2014) and the small J-tipped GW group (from March 2014 to September 2016).Difficulty in the endoscopic approach Previous gastrectomy with Billroth II or Roux-en-Y reconstructionPrevious papillectomyApproach from the minor papillaPrecuttingRecent history of acute pancreatitisUse of other types of GWs for selective pancreatic cannulation instead of small J-tipped or angle-tipped GWsUse of DiclofenacThe following cases were excluded and only the first treatment was analyzed for overlapping cases:This is a retrospective observational study at a single center.Angle-tipped GWs  or small J-tipped GWs  were used for insertion into the pancreatic duct (first GW) . Both tyThe procedures were carried out using side-viewing duodenoscopes  and a standard injection catheter . Neutral positions of each GWs for cannulation to the pancreatic duct were determined as follows: the angle-tipped GW was used aligned with the tip of catheter, and the small J-tipped GW was used with the J shape of the tip .Selective pancreatic cannulation was conducted using the contrast method. All endoscopic procedures were performed by dedicated ERCP training fellow under directly supervising senior endoscopists who had more than 1000 ERCP experiences. Eight trainees in the angle-tipped GW group and 10 trainees in the small J-tipped GW group performed endoscopic intervention, but the 2 trainers who supervising them were common in both groups.All procedures were performed under conscious sedation using intravenous midazolam (3–10 mg) and pentazocine (15 mg). Scopolamine butylbromide (20 mg) or glucagon (1 mg) was intravenously administered to control the intestinal peristaltic motion. To prevent PEP, ulinastatin  was intravenously administered immediately after treatment.(1) Assessment of Efficacy. The first GW procedure completion rate and final procedure completion rate were compared between the small J-tipped and angle-tipped GW groups. An analysis was also conducted on factors related to the first GW procedure completion.(2) Assessment of Safety. The post-ERCP complication rate and severity were compared between the small J-tipped and angle-tipped GW groups. A diagnosis of PEP was made on the presence of continuous abdominal pain at least 24 h after procedure and an increase in serum amylase level greater than three times the upper normal limit. Severity was classified according to the modified Cotton's criteria [criteria  with theAn analysis was also performed pertaining to the risk factors for PEP.The chi-squared test or the Fisher's exact test was used for a univariate analysis of category data, with the Student's t-test used for the analysis of quantitative data. In assessing the first GW procedure completion-related factors and PEP risk factors, a multivariate analysis was performed using multiple logistic regression analysis, which was conducted after univariate analysis using factor P < 0.20. The level of statistical significance was set at P < 0.05.In total, 377 patients underwent pancreatic duct endoscopic intervention between January 2011 and September 2016. After excluding patients who met any of the exclusion criteria, we enrolled 114 patients in the small J group and 180 patients in the angle group .Selective pancreatic cannulation succeeded in 109 (96%) cases in the small J-tipped GW group and 177 (98%) cases in the angle-tipped GW group .  Table Although pancreatic duct endoscopic intervention is performed with a GW indwelling in the pancreatic duct, the insertion of the GW is difficult because the pancreatic duct is anatomically complicated with advanced curves and strictures. In recent years, J-tipped GWs to be used for luminal passage and stricture penetration have been developed and reported to be beneficial in selective biliary cannulation. Although the J-shaped tip is considered to be more suitable for pancreatic intervention owing to the low risks of damaging the pancreatic duct and wrong insertion into a branch duct, the utility and safety of J-shaped tip GW in pancreatic intervention had not previously been examined . Here, tThe major problem associated with pancreatic duct endoscopic intervention is postoperative pancreatitis. GW insertion into the pancreatic duct is considered to be a risk factor for pancreatitis and injury of the pancreatic duct caused by the GW may result in serious outcomes –6, 11. ASakai et al. have reported on the preventive effect of loop-tipped GWs against PEP . Loop-tiThis study had several limitations. First, it had a retrospective design and there were intergroup differences in background diseases and pancreatic duct findings . Although these factors may have affected efficacy and safety as bias factors, it is evident that the utility of small J-tipped GWs is high because chronic pancreatitis and acute-angled pancreatic duct are rather difficult procedure-related factors . In addiIn conclusion, present study suggested that small J-tipped GWs not only offered better usefulness than angle-tipped GWs in pancreatic duct endoscopic intervention but also reduce the risk of PEP.Small J-tipped GWs increase the success rate of the pancreatic intervention as well as a reduced risk of developing postoperative pancreatitis.


The incidence of central nervous system (CNS) metastases in breast cancer patients is rising and has become a major clinical challenge. Only few data are published concerning risk factors for the development of CNS metastases as a first site of metastatic disease in breast cancer patients. Moreover, the incidence of CNS metastases after modern neoadjuvant treatment is not clear.n = 3160) where patients received targeted treatment in addition to taxane and anthracycline-based chemotherapy.We analyzed clinical factors associated with the occurrence of CNS metastases as the first site of metastatic disease in breast cancer patients after neoadjuvant treatment in the trials GeparQuinto and GeparSixto  of a total of 3160 patients developed CNS metastases as the first site of recurrence and 411 (13%) patients had metastatic disease outside the CNS. Thirty-six patients (1%) developed both CNS metastases and other distant metastases as the first site of metastatic disease. Regarding subtypes of the primary tumor, 1% of luminal A-like (11/954), 2% of luminal B-like (7/381), 4% of HER2-positive (34/809), and 6% of triple-negative patients (56/1008) developed CNS metastases as the first site of metastatic disease.p = 0.021), node-positive disease , no pCR after neoadjuvant chemotherapy , and HER2-positive  or triple-negative subtype .In multivariate analysis, risk factors for the development of CNS metastases were larger tumor size (cT3–4; HR 1.63, 95% CI 1.08–2.46, Especially patients with HER2-positive and triple-negative tumors are at risk of developing CNS metastases despite effective systemic treatment. A better understanding of the underlying mechanisms is required in order to develop potential preventive strategies. Central nervous system (CNS) metastases in breast cancer patients are a clinically relevant problem. CNS metastases are associated with shorter survival and impaired quality of life compared with extracranial metastases. Despite the use of neurosurgery and radiotherapy, only the minority of patients survives longer than 1 year . Pan = 115) .A different probability of breast cancer subtypes to metastasize into the brain was also described by others –20. Sonin = 9) or TNBC (n = 7) primary tumor.A noteworthy finding in our study relates to pCR rates in the analyzed subgroups. The pCR rate in patients with CNS metastases as the first site of metastatic disease was higher (15%) compared to patients with other distant metastases as the first site of metastatic disease (9%). Thus, a good response rate to a systemic treatment seems not to have a deciding role in the prevention of CNS metastases. Possibly, more aggressive disseminated tumor cells, which also did not respond to a systemic treatment, gain a propensity to overcome the blood-brain barrier and survive in the central nervous system. Most systemic treatment approaches are assumed not to cross the intact blood-brain barrier; this fact might explain why tumor cells that already reached the CNS cannot be affected. Probably, the initially more aggressive tumor is also a significant risk factor in the development of CNS metastases. In our cohort, 16 patients developed CNS metastases as the first site of metastatic disease despite achieving a pCR. All of them had either HER2-positive (The strength of our analysis is a large cohort of patients who received up-to-date treatment with anthracyclines and taxanes as well as up-to-date HER2-directed therapy. However, given the relatively low incidence of CNS metastases, we cannot identify differences between the incidence of CNS metastases in different treatment groups of the trials, e.g., between lapatinib- and trastuzumab-treated patients with HER2-positive breast cancer or in the cohort of triple-negative patients with carboplatin therapy compared to patients without carboplatin therapy. A small number of patients with an available BRCA status in our cohort also do not allow a confident statistical statement concerning the correlation with the development of CNS metastases and may be regarded solely as trend. A further limitation of our analysis is that the incidence of CNS metastases after the occurrence of metastatic disease was not documented, as in many other trials. This point must be taken into consideration by the interpretation of Fig. In conclusion, our analysis showed that especially patients with HER2-positive and triple-negative tumors are at risk of developing CNS metastases despite active systemic treatment, especially when no pCR was reached. However, also patients with pCR are at risk of developing CNS metastases. A better understanding of the underlying mechanisms is required in order to develop potential preventive strategies and should help to identify molecular mechanisms of resistance to modern therapies and clonal selection of tumor cells with special brain tropism. Further studies should aim to identify a group of patients that might have a benefit of screening for brain metastases. Further research on biomarkers for CNS metastasis development should be performed. A translational research project in our cohort is ongoing.