Design of small-molecule active-site inhibitors of the S1A family proteases as procoagulant and anticoagulant drugs

Vitamin K antagonists (VKA) have long been the default drugs for anticoagulant management in venous thrombosis. While efficacious, they are difficult to use due to interpatient dose–response variability and the risks of bleeding. The approval of fondaparinux, a heparin-derived factor Xa (fXa) inhibitor, provided validation for the development of direct oral anticoagulants (DOAC), and currently such inhibitors of thrombin and fXa are in clinical use. These agents can be used without regular coagulation monitoring, but the inherent risk of bleeding complications associated with blocking the common coagulation pathway remains. Efforts are now underway to develop DOACs that inhibit components of the intrinsic and extrinsic coagulation cascades upstream of thrombin and fX. Evidence from humans and from transgenic animal models suggests that this strategy may provide a better therapeutic margin between antithrombotic and antihemostatic effects. Here the design of active-site inhibitors of S1A proteases involved in coagulation and fibrinolysis is summarized

RNA silencing can explain chlorotic infection patterns on plant leaves

<p>Abstract</p> <p>Background</p> <p>RNA silencing has been implicated in virus symptom development in plants. One common infection symptom in plants is the formation of chlorotic tissue in leaves. Chlorotic and healthy tissue co-occur on a single leaf and form patterns. It has been shown that virus levels in chlorotic tissue are high, while they are low in healthy tissue. Additionally, the presence of siRNAs is confined to the chlorotic spots and the boundaries between healthy and infected tissue. These results strongly indicate that the interaction between virus growth and RNA silencing plays a role in the formation of infection patterns on leaves. However, how RNA silencing leads to the intricate patterns is not known.</p> <p>Results</p> <p>Here we elucidate the mechanisms leading to infection patterns and the conditions which lead to the various patterns observed. We present a modeling approach in which we combine intra- and inter-cellular dynamics of RNA silencing and viral growth. We observe that, due to the spread of viruses and the RNA silencing response, parts of the tissue become infected while other parts remain healthy. As is observed in experiments high virus levels coincide with high levels of siRNAs, and siRNAs are also present in the boundaries between infected and healthy tissue. We study how single- and double-stranded cleavage by Dicer and amplification by RNA-dependent RNA polymerase can affect the patterns formed.</p> <p>Conclusion</p> <p>This work shows that RNA silencing and virus growth within a cell, and the local spread of virions and siRNAs between cells can explain the heterogeneous spread of virus in leaf tissue, and therewith the observed infection patterns in plants.</p

Influence of viral genes on the cell-to-cell spread of RNA silencing

The turnip crinkle virus-based vector TCV–GFPΔCP had been devised previously to study cell-to-cell and long-distance spread of virus-induced RNA silencing. TCV–GFPΔCP, which had been constructed by replacing the coat protein (CP) gene with a green fluorescent protein (GFP) coding sequence, was able to induce RNA silencing in single epidermal cells, from which RNA silencing spread from cell-to-cell. Using this unique local silencing assay together with mutagenesis analysis, two TCV genes, p8 and p9, which were involved in the intercellular spread of virus-induced RNA silencing, were identified. TCV–GFPΔCP and its p8- or p9-mutated derivatives, TCVmp8–GFPΔCP and TCVmp9–GFPΔCP, replicated efficiently but were restricted to single Nicotiana benthamiana epidermal cells. TCV–GFPΔCP, TCVmp8–GFPΔCP, or TCVmp9–GFPΔCP was able to initiate RNA silencing that targeted and degraded recombinant viral RNAs in inoculated leaves of the GFP-expressing N. benthamiana line 16c. However, cell-to-cell spread of silencing to form silencing foci was triggered only by TCV–GFPΔCP. Non-replicating TCVmp88–GFPΔCP and TCVmp28mp88–GFPΔCP with dysfunctional replicase genes, and single-stranded gfp RNA did not induce RNA silencing. Transient expression of the TCV p9 protein could effectively complement TCVmp9–GFPΔCP to facilitate intercellular spread of silencing. These data suggest that the plant cellular trafficking machinery could hijack functional viral proteins to permit cell-to-cell movement of RNA silencing

Involvement of RDR6 in short-range intercellular RNA silencing in Nicotiana benthamiana

In plants, non-cell autonomous RNA silencing spreads between cells and over long distances. Recent work has revealed insight on the genetic and molecular components essential for cell-to-cell movement of RNA silencing in Arabidopsis. Using a local RNA silencing assay, we report on a distinct mechanism that may govern the short-range (6–10 cell) trafficking of virus-induced RNA silencing from epidermal to neighbouring palisade and spongy parenchyma cells in Nicotiana benthamiana. This process involves a previously unrecognised function of the RNA-dependent RNA polymerase 6 (RDR6) gene. Our data suggest that plants may have evolved distinct genetic controls in intercellular RNA silencing among different types of cells

Graft-Transmitted siRNA Signal from the Root Induces Visual Manifestation of Endogenous Post-Transcriptional Gene Silencing in the Scion

In plants, post-transcriptional gene silencing (PTGS) spreads systemically, being transmitted from the silenced stock to the scion expressing the corresponding transgene. It has been reported that a graft-transmitted siRNA signal can also induce PTGS of an endogenous gene, but this was done by top-grafting using silenced stock. In the present study involving grafting of Nicotiana benthamiana, we found that PTGS of an endogenous gene, glutamate-1-semialdehyde aminotransferase (GSA), which acts as a visible marker of RNAi via inhibition of chlorophyll synthesis, was manifested along the veins of newly developed leaves in the wild-type scion by the siRNA signal synthesized only in companion cells of the rootstock

Non-suicidal reasons for self-harm: a systematic review of self-reported accounts

Background: Self-harm is a major public health problem yet current healthcare provision is widely regarded as inadequate. One of the barriers to effective healthcare is the lack of a clear understanding of the functions self-harm may serve for the individual. The aim of this review is to identify first-hand accounts of the reasons for self-harm from the individual's perspective. Method: A systematic review of the literature reporting first-hand accounts of the reasons for self-harm other than intent to die. A thematic analysis and ‘best fit' framework synthesis was undertaken to classify the responses. Results: The most widely researched non-suicidal reasons for self-harm were dealing with distress and exerting interpersonal influence. However, many first-hand accounts included reasons such as self-validation, and self-harm to achieve a personal sense of mastery, which suggests individuals thought there were positive or adaptive functions of the act not based only on its social effects. Limitations: Associations with different sub-population characteristics or with the method of harm were not available from most studies included in the analysis. Conclusions: Our review identified a number of themes that are relatively neglected in discussions about self-harm, which we summarised as self-harm as a positive experience and defining the self. These self-reported “positive” reasons may be important in understanding and responding especially to repeated acts of self-harm

APC/C-Mediated Degradation of dsRNA-Binding Protein 4 (DRB4) Involved in RNA Silencing

Background: Selective protein degradation via the ubiquitin-26S proteasome is a major mechanism underlying DNA replication and cell division in all Eukaryotes. In particular, the APC/C (Anaphase Promoting Complex or Cyclosome) is a master ubiquitin protein ligase (E3) that targets regulatory proteins for degradation allowing sister chromatid separation and exit from mitosis. Interestingly, recent work also indicates that the APC/C remains active in differentiated animal and plant cells. However, its role in post-mitotic cells remains elusive and only a few substrates have been characterized. Methodology/Principal Findings: In order to identify novel APC/C substrates, we performed a yeast two-hybrid screen using as the bait Arabidopsis APC10/DOC1, one core subunit of the APC/C, which is required for substrate recruitment. This screen identified DRB4, a double-stranded RNA binding protein involved in the biogenesis of different classes of small RNA (sRNA). This protein interaction was further confirmed in vitro and in plant cells. Moreover, APC10 interacts with DRB4 through the second dsRNA binding motif (dsRBD2) of DRB4, which is also required for its homodimerization and binding to its Dicer partner DCL4. We further showed that DRB4 protein accumulates when the proteasome is inactivated and, most importantly, we found that DRB4 stability depends on APC/C activity. Hence, depletion of Arabidopsis APC/C activity by RNAi leads to a strong accumulation of endogenous DRB4, far beyond its normal level of accumulation. However, we could not detect any defects in sRNA production in lines where DRB4 was overexpressed

Evidence for Antisense Transcription Associated with MicroRNA Target mRNAs in Arabidopsis

Antisense transcription is a pervasive phenomenon, but its source and functional significance is largely unknown. We took an expression-based approach to explore microRNA (miRNA)-related antisense transcription by computational analyses of published whole-genome tiling microarray transcriptome and deep sequencing small RNA (smRNA) data. Statistical support for greater abundance of antisense transcription signatures and smRNAs was observed for miRNA targets than for paralogous genes with no miRNA cleavage site. Antisense smRNAs were also found associated with MIRNA genes. This suggests that miRNA-associated “transitivity” (production of small interfering RNAs through antisense transcription) is more common than previously reported. High-resolution (3 nt) custom tiling microarray transcriptome analysis was performed with probes 400 bp 5′ upstream and 3′ downstream of the miRNA cleavage sites (direction relative to the mRNA) for 22 select miRNA target genes. We hybridized RNAs labeled from the smRNA pathway mutants, including hen1-1, dcl1-7, hyl1-2, rdr6-15, and sgs3-14. Results showed that antisense transcripts associated with miRNA targets were mainly elevated in hen1-1 and sgs3-14 to a lesser extent, and somewhat reduced in dcl11-7, hyl11-2, or rdr6-15 mutants. This was corroborated by semi-quantitative reverse transcription PCR; however, a direct correlation of antisense transcript abundance in MIR164 gene knockouts was not observed. Our overall analysis reveals a more widespread role for miRNA-associated transitivity with implications for functions of antisense transcription in gene regulation. HEN1 and SGS3 may be links for miRNA target entry into different RNA processing pathways