Design of dual ligands using excessive pharmacophore query alignment

Dual- or multi-target ligands have gained increased attention in the past years due to several advantages, including more simple pharmacokinetic and phamarcodynamic properties compared to a combined application of several drugs. Furthermore multi-target ligands often possess improved efficacy. We present a new approach for the discovery of dual-target ligands using aligned pharmacophore models combined with a shape-based scoring. Starting with two sets of known active compounds for each target, a number of different pharmacophore models is generated and subjected to pairwise graph-based alignment using the Kabsch-Algorithm. Since a compound may be able to bind to different targets in different conformations, the algorithm aligns pairs of pharmacophore models sharing the same features which are not necessarily at the exactly same spatial distance. Using the aligned models, a pharmacophore search on a multi-conformation-database is performed to find compounds matching both models. The potentially “dual” ligands are scored by a shape-based comparison with the known active molecules using ShaEP. Using this approach, we performed a prospective fragment-based virtual screening for dual 5-LO/sEH inhibitors. Both enzymes play an important role in the arachidonic acid cascade and are involved in inflammatory processes, pain, cardiovascular diseases and allergic reactions. Beside several new selective inhibitors we were able to find a compound inhibiting both enzymes in low micromolar concentrations. The results indicate that the idea of aligned pharmacophore models can be successfully employed for the discovery of dual-target ligands

Circulating markers of arterial thrombosis and late-stage age-related macular degeneration: a case-control study.

PURPOSE: The aim of this study was to examine the relation of late-stage age-related macular degeneration (AMD) with markers of systemic atherothrombosis. METHODS: A hospital-based case-control study of AMD was undertaken in London, UK. Cases of AMD (n=81) and controls (n=77) were group matched for age and sex. Standard protocols were used for colour fundus photography and to classify AMD; physical examination included height, weight, history of or treatment for vascular-related diseases and smoking status. Blood samples were taken for measurement of fibrinogen, factor VIIc (FVIIc), factor VIIIc, prothrombin fragment F1.2 (F1.2), tissue plasminogen activator, and von Willebrand factor. Odds ratios from logistic regression analyses of each atherothrombotic marker with AMD were adjusted for age, sex, and established cardiovascular disease risk factors, including smoking, blood pressure, body mass index, and total cholesterol. RESULTS: After adjustment FVIIc and possibly F1.2 were inversely associated with the risk of AMD; per 1 standard deviation increase in these markers the odds ratio were, respectively, 0.62 (95% confidence interval 0.40, 0.95) and 0.71 (0.46, 1.09). None of the other atherothrombotic risk factors appeared to be related to AMD status. There was weak evidence that aspirin is associated with a lower risk of AMD. CONCLUSIONS: This study does not provide strong evidence of associations between AMD and systematic markers of arterial thrombosis, but the potential effects of FVIIc, and F1.2 are worthy of further investigation

Enhancer RNA Transcription Is Essential for a Novel CSF1 Enhancer in Triple-Negative Breast Cancer

Enhancers are critical regulatory elements in the genome that help orchestrate spatiotemporal patterns of gene expression during development and normal physiology. In cancer, enhancers are often rewired by various genetic and epigenetic mechanisms for the activation of oncogenes that lead to initiation and progression. A key feature of active enhancers is the production of non-coding RNA molecules called enhancer RNAs, whose functions remain unknown but can be used to specify active enhancers de novo. Using a combination of eRNA transcription and chromatin modifications, we have identified a novel enhancer located 30 kb upstream of Colony Stimulating Factor 1 (CSF1). Notably, CSF1 is implicated in the progression of breast cancer, is overexpressed in triple-negative breast cancer (TNBC) cell lines, and its enhancer is primarily active in TNBC patient tumors. Genomic deletion of the enhancer (via CRISPR/Cas9) enabled us to validate this regulatory element as a bona fide enhancer of CSF1 and subsequent cell-based assays revealed profound effects on cancer cell proliferation, colony formation, and migration. Epigenetic silencing of the enhancer via CRISPR-interference assays (dCas9-KRAB) coupled to RNA-sequencing, enabled unbiased identification of additional target genes, such as RSAD2, that are predictive of clinical outcome. Additionally, we repurposed the RNA-guided RNA-targeting CRISPR-Cas13 machinery to specifically degrade the eRNAs transcripts produced at this enhancer to determine the consequences on CSF1 mRNA expression, suggesting a post-transcriptional role for these non-coding transcripts. Finally, we test our eRNA-dependent model of CSF1 enhancer function and demonstrate that our results are extensible to other forms of cancer. Collectively, this work describes a novel enhancer that is active in the TNBC subtype, which is associated with cellular growth, and requires eRNA transcripts for proper enhancer function. These results demonstrate the significant impact of enhancers in cancer biology and highlight their potential as tractable targets for therapeutic intervention

Application of new methods of environment analysis and assessment in landscape audits : case studies of urban areas like Czestochowa, Poland

Following the 2000 European Landscape Convention, a new act strengthening landscape protection instruments has been in force since 2015. It sets forth legal aspects of landscape shaping (Dziennik Ustaw 2015, poz. 774) and introduces landscape audits at the province level. A landscape audit consists in identification and characterization of selected landscapes, assessment of their value, selection of so-called priority landscapes and identification of threats for preservation of their value. An audit complies with GIS standards. Analyses use source materials, i.e. digital maps of physical-geographical mesoregions, current topographic maps of digital resources of cartographic databases, latest orthophotomaps and DTMs, maps of potential vegetation, geobotanic regionalization, historic-cultural regionalization and natural landscape types, documentation of historical and cultural values and related complementary resources. A special new methodology (Solon et al. 2014), developed for auditing, was tested in 2015 in an urban area (Myga-Piatek et al. 2015). Landscapes are characterized by determining their analytic (natural and cultural) and synthetic features, with particular focus on the stage of delimitation and identification of landscape units in urban areas. Czestochowa was selected as a case study due to its large natural (karst landscapes of the Czestochowa Upland, numerous forests, nature reserves) and cultural (Saint Mary’s Sanctuary, unique urban architecture) potential. Czestochowa is also a city of former iron ore and mineral resources exploitation, still active industry, dynamic urban sprawl within former farming areas, and dynamically growing tourism. Landscape delimitation and identification distinguished 75 landscape units basing on uniform landscape background (uniform cover and use of the land). Landscape assessment used a new assessment method for anthropogenic transformation of landscape – the indicator describing the correlation between the mean shape index (MSI) and the Shannon diversity index (SHDI) (Pukowiec-Kurda, Sobala 2016). Particular threats and planning suggestions, useful in development of urban areas, were presented for selected priority landscapes

Why whales are big but not bigger : physiological drivers and ecological limits in the age of ocean giants

This research was funded in part by grants from the National Science Foundation (IOS-1656676, IOS-1656656; OPP-1644209 and 07-39483), the Office of Naval Research (N000141612477), and a Terman Fellowship from Stanford University. All procedures in USA were conducted under approval of the National Marine Fisheries Service (Permits 781-1824, 16163, 14809, 16111, 19116, 15271, 20430), Canada DFO SARA/MML 2010-01/SARA-106B, National Marine Sanctuaries (MULTI-2017-007), Antarctic Conservation Act (2009-014, 2015-011) and institutional IACUC committee protocols. Fieldwork, data collection and data processing for M. densirostris were funded by the Office of Naval Research grants N00014-07-10988, N00014-07-11023, N00014-08-10990, N00014-18-1-2062, and 00014-15-1-2553, and the U.S. Strategic Environmental Research and Development Program Grant SI-1539. PLT gratefully acknowledges funding from funding the MASTS pooling initiative (The Marine Alliance for Science and Technology for Scotland). MASTS is funded by the Scottish Funding Council (HR09011) and contributing institutions.The largest animals are marine filter feeders, but the underlying mechanism of their large size remains unexplained. We measured feeding performance and prey quality to demonstrate how whale gigantism is driven by the interplay of prey abundance and harvesting mechanisms that increase prey capture rates and energy intake. The foraging efficiency of toothed whales that feed on single prey is constrained by the abundance of large prey, whereas filter-feeding baleen whales seasonally exploit vast swarms of small prey at high efficiencies. Given temporally and spatially aggregated prey, filter feeding provides an evolutionary pathway to extremes in body size that are not available to lineages that must feed on one prey at a time. Maximum size in filter feeders is likely constrained by prey availability across space and time.PostprintPeer reviewe

The Crystal Structure of the Human Co-Chaperone P58IPK

P58IPK is one of the endoplasmic reticulum- (ER-) localised DnaJ (ERdj) proteins which interact with the chaperone BiP, the mammalian ER ortholog of Hsp70, and are thought to contribute to the specificity and regulation of its diverse functions. P58IPK, expression of which is upregulated in response to ER stress, has been suggested to act as a co-chaperone, binding un- or misfolded proteins and delivering them to BiP. In order to give further insights into the functions of P58IPK, and the regulation of BiP by ERdj proteins, we have determined the crystal structure of human P58IPK to 3.0 Å resolution using a combination of molecular replacement and single wavelength anomalous diffraction. The structure shows the human P58IPK monomer to have a very elongated overall shape. In addition to the conserved J domain, P58IPK contains nine N-terminal tetratricopeptide repeat motifs, divided into three subdomains of three motifs each. The J domain is attached to the C-terminal end via a flexible linker, and the structure shows the conserved Hsp70-binding histidine-proline-aspartate (HPD) motif to be situated on the very edge of the elongated protein, 100 Å from the putative binding site for unfolded protein substrates. The residues that comprise the surface surrounding the HPD motif are highly conserved in P58IPK from other organisms but more varied between the human ERdj proteins, supporting the view that their regulation of different BiP functions is facilitated by differences in BiP-binding

Acoustic risk balancing by marine mammals : anthropogenic noise can influence the foraging decisions by seals

Funding: Natural Environment Research Council (Grant Number(s): NE/J004251/1, NE/R015007/1)1. Avoidance of anthropogenic sounds has been measured in many species. The results, which are typically based on observations in limited exposure contexts, are frequently used to inform policy and the regulation of industrial activities. However, the occurrence and magnitude of avoidance may be a consequence of complex risk-balancing decisions made by animals. The importance of the factors in decision-making, such as perceived risks associated with the sounds or prey quantity and quality during sound exposure, is unknown. 2. Here we address this knowledge gap by measuring the relative influence of perceived –risk of a sound (silence, pile driving, and a tidal turbine) and prey patch quality on decision-making and foraging success in grey seals Halichoerus grypus. 3. Seals were given access to two underwater ‘prey patches’ in an experimental pool where fish were delivered at controlled rates to simulate a low-density (LD) and a high-density (HD) prey patch. Acoustic playbacks were made using an underwater speaker above one of the prey patches (randomised during the study), and three decision and foraging metrics (foraging duration, foraging effort allocation between the prey patches, and foraging success) were measured. 4. Foraging success was highest during silent controls and was similar regardless of speaker location (LD/HD). Under the tidal turbine and pile-driving treatments, foraging success was similar to the controls when the speaker was located at the HD prey patch but was significantly reduced (~16%–28% lower) when the speaker was located at the LD prey patch. Foraging decisions by the seals were consistent with a risk/profit balancing approach. Avoidance rates depend on the quality of the prey patch as well as the perceived risk. 5. Policy implications. The results suggest that foraging context is important when interpreting avoidance behaviour and should be considered when predicting the effects of anthropogenic activities. For example, sound exposure in different prey patch qualities may result in markedly different avoidance behaviour, potentially leading to contrasting predictions of impact in Environmental Assessments. We recommend future studies explicitly consider foraging context, and other contextual factors such as behavioural state (e.g. foraging or travelling) and habitat quality.Publisher PDFPeer reviewe

Crystal Structures of the ATPase Domains of Four Human Hsp70 Isoforms: HSPA1L/Hsp70-hom, HSPA2/Hsp70-2, HSPA6/Hsp70B', and HSPA5/BiP/GRP78

The 70-kDa heat shock proteins (Hsp70) are chaperones with central roles in processes that involve polypeptide remodeling events. Hsp70 proteins consist of two major functional domains: an N-terminal nucleotide binding domain (NBD) with ATPase activity, and a C-terminal substrate binding domain (SBD). We present the first crystal structures of four human Hsp70 isoforms, those of the NBDs of HSPA1L, HSPA2, HSPA5 and HSPA6. As previously with Hsp70 family members, all four proteins crystallized in a closed cleft conformation, although a slight cleft opening through rotation of subdomain IIB was observed for the HSPA5-ADP complex. The structures presented here support the view that the NBDs of human Hsp70 function by conserved mechanisms and contribute little to isoform specificity, which instead is brought about by the SBDs and by accessory proteins.This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1

Uncoupling of sexual reproduction from homologous recombination in homozygous Oenothera species

Salient features of the first meiotic division are independent segregation of chromosomes and homologous recombination (HR). In non-sexually reproducing, homozygous species studied to date HR is absent. In this study, we constructed the first linkage maps of homozygous, bivalent-forming Oenothera species and provide evidence that HR was exclusively confined to the chromosome ends of all linkage groups in our population. Co-segregation of complementary DNA-based markers with the major group of AFLP markers indicates that HR has only a minor role in generating genetic diversity of this taxon despite its efficient adaptation capability. Uneven chromosome condensation during meiosis in Oenothera may account for restriction of HR. The use of plants with ancient chromosomal arm arrangement demonstrates that limitation of HR occurred before and independent from species hybridizations and reciprocal translocations of chromosome arms—a phenomenon, which is widespread in the genus. We propose that consecutive loss of HR favored the evolution of reciprocal translocations, beneficial superlinkage groups and ultimately permanent translocation heterozygosity

Characterization and Whole Genome Analysis of Human Papillomavirus Type 16 E1-1374^63nt Variants

Background. The variation of the most common Human papillomavirus (HPV) type found in cervical cancer, the HPV16, has been extensively investigated in almost all viral genes. The E1 gene variation, however, has been rarely studied. The main objective of the present investigation was to analyze the variability of the E6 and E1 genes, focusing on the recently identified E1-1374^63nt variant. Methodology/Principal Findings. Variation within the E6 of 786 HPV16 positive cervical samples was analyzed using high-resolution melting, while the E1-1374^63nt duplication was assayed by PCR. Both techniques were supplemented with sequencing. The E1-1374^63nt duplication was linked with the E-G350 and the E-C109/G350 variants. In comparison to the referent HPV16, the E1-1374^63nt E-G350 variant was significantly associated with lower grade cervical lesions (p=0.029), while the E1-1374^63nt E-C109/G350 variant was equally distributed between high and low grade lesions. The E1-1374^63nt variants were phylogenetically closest to E-G350 variant lineage (A2 sub-lineage based on full genome classification). The major differences between E1-1374^63nt variants were within the LCR and the E6 region. On the other hand, changes within the E1 region were the major differences from the A2 sub-lineage, which has been historically but inconclusively associated with high grade cervical disease. Thus, the shared variations cannot explain the particular association of the E1-1374^63nt variant with lower grade cervical lesions. Conclusions/Significance. The E1 region has been thus far considered to be well conserved among all HPVs and therefore uninteresting for variability studies. However, this study shows that the variations within the E1 region could possibly affect cervical disease, since the E1-1374^63nt E-G350 variant is significantly associated with lower grade cervical lesions, in comparison to the A1 and A2 sub-lineage variants. Furthermore, it appears that the silent variation 109T>C of the E-C109/G350 variant might have a significant role in the viral life cycle and warrants further study