Novel bi- and trifunctional inhibitors of tumor-associated proteolytic systems

Serine proteases, cysteine proteases, and matrix metalloproteinases (MMPs) are involved in cancer cell invasion and metastasis. Recently, a recombinant bifunctional inhibitor (chCysuPA(19-31)) directed against cysteine proteases and the urokinasetype plasminogen activator (uPA)/plasmin serine protease system was generated by introducing the uPA receptor (uPAR)binding site of uPA into chicken cystatin (chCysWT). In the present study, we designed and recombinantly produced multifunctional inhibitors also targeting MMPs. The inhibitors comprise the Nterminal inhibitory domain of human TIMP-1 (tissue inhibitor of matrix metalloproteinase-1) or TIMP-3, fused to chCysuPA(19-31) or chCysWT. As demonstrated by various techniques, these fusion proteins effectively interfere with all three targeted protease systems. In in vitro Matrigel invasion assays, the addition of recombinant inhibitors strongly reduced invasion of ovarian cancer cells (OVMZ-6\#8). Additionally, OVMZ 6\#8 cells were stably transfected with expression plasmids encoding the various inhibitors. Synthesis and secretion of the inhibitors was verified by a newly developed ELISA, which selectively detects the recombinant proteins. Invasive capacity of inhibitorproducing cells was significantly reduced compared to vectortransfected control cells. Thus, these novel, compact, and smallsize inhibitors directed against up to three different tumorassociated proteolytic systems may represent promising agents for prevention of tumor cell migration and metastasis

High Resolution X-ray Spectra of RS Ophiuchi (2006 and 2021): Revealing the cause of SSS variability

Context. The ∼ 10 − 20 year recurrent symbiotic nova RS Oph exploded on 2021 August 9, the seventh confirmed recorded outburst since 1898. During the previous outburst in 2006, the current fleet of X-ray space observatories was already in operation, and thanks to the longevity of Swift, XMM-Newton, and Chandra, a direct comparison between these two outbursts is possible. The Swift monitoring campaign revealed similar behaviour during the early shock phase but very different behaviour during the super-soft source (SSS) phase. Two XMM-Newton⋆ observations were made during the 2021 SSS phase on days 37.1 and 55.6 after the 2021 optical peak. We focus in this work on the bright SSS observation on day 55.6 and compare to SSS Chandra and XMM-Newton grating observations made on days 39.7, 54, and 66.9 after the 2006 optical peak. Aims. By exploring the reasons for the differences between the 2006 and 2021 outbursts, we aim to obtain a better general understanding of the emission and absorption mechanisms. While the emission mechanisms hold the key to the physics of novae and nuclear burning, absorption processes may dominate what we observe, and we aim to explore the cause of the gross initial variability in the observed SSS emission. Methods. We present a novel approach to down-scaling the observed (brighter) 2006 SSS spectra to match the 2021 day 55.6 spectrum by parameter optimisation of: (1) a constant factor (representing fainter source emission, smaller radius, eclipses, etc.), (2) a multi-ionisation photoelectric absorption model (representing different line-of-sight absorption), and (3) scaling with a ratio of two blackbody models with different effective temperatures (representing different brightness and colours). This model approach does not depend on a source model assuming the intrinsic source to be the same. It is therefore more sensitive to incremental changes than modelling approaches where source and absorption are modelled simultaneously. Results. The 2021d55.6 spectrum can be reproduced remarkably well by multiplying the (brighter) 2006d39.7 and 2006d54 spectra with the absorption model, while the 2006d66.9 spectrum requires additional colour changes to match the 2021.d55.6 spectrum. The 2006d39.7 spectrum much more closely resembles the 2021d55.6 spectrum in shape and structure than the same-epoch 2006d54 spectrum: The spectra on days 2006d39.7 and 2021d55.6 are richer in absorption lines with a deeper O i absorption edge, and blueshifts are higher (∼ 1200 km s−1 ) than on day 2006d54 (∼ 700 km s−1 ). In the SSS light curves on days 2006d39.7, 2006d54, and 2021d55.6, brightness and hardness variations are correlated, indicating variations of the O i column density. Only on day 2006d39.7, a 1000s lag is observed. The 35s period was detected on day 2021d55.6 with lower significance compared to 2006d54. Conclusions. We conclude that the central radiation source is the same, while absorption is the principal reason for observing lower soft-X-ray emission in 2021 than in 2006. This is consistent with a similar 2006 and 2021 [Fe x] line-flux evolution. We explain the reduction in line blueshift, depth in O i edge, and number of absorption lines from day 2006d39.7 to 2006d54 by deceleration and heating of the ejecta within the stellar wind of the companion. In 2021, less such deceleration and heating was observed, which we interpret as due to viewing at different angles through an inhomogeneous density distribution of the stellar wind, allowing free expansion in some directions (probed in 2021) and a higher degree of deceleration in others (probed in 2006). The higher absorption in 2021 can then be explained by the lower-temperature absorbing plasma being more opaque to soft X-rays. Our approach of scaling observations against observations is free of ambiguities from imperfect source models and can be applied to other grating spectra with complex continuum sources

Combined mRNA expression levels of members of the urokinase plasminogen activator (uPA) system correlate with disease-associated survival of soft-tissue sarcoma patients

<p>Abstract</p> <p>Background</p> <p>Members of the urokinase-type plasminogen activator (uPA) system are up-regulated in various solid malignant tumors. High antigen levels of uPA, its inhibitor PAI-1 and its receptor uPAR have recently been shown to be associated with poor prognosis in soft-tissue sarcoma (STS) patients. However, the mRNA expression of uPA system components has not yet been comprehensively investigated in STS patients.</p> <p>Methods</p> <p>The mRNA expression level of uPA, PAI-1, uPAR and an uPAR splice variant, uPAR-del4/5, was analyzed in tumor tissue from 78 STS patients by quantitative PCR.</p> <p>Results</p> <p>Elevated mRNA expression levels of PAI-1 and uPAR-del4/5 were significantly associated with clinical parameters such as histological subtype (<it>P </it>= 0.037 and <it>P </it>< 0.001, respectively) and higher tumor grade (<it>P </it>= 0.017 and <it>P </it>= 0.003, respectively). In addition, high uPAR-del4/5 mRNA values were significantly related to higher tumor stage of STS patients (<it>P </it>= 0.031). On the other hand, mRNA expression of uPA system components was not significantly associated with patients' survival. However, in STS patients with complete tumor resection (R0), high PAI-1 and uPAR-del4/5 mRNA levels were associated with a distinctly increased risk of tumor-related death (RR = 6.55, <it>P </it>= 0.054 and RR = 6.00, <it>P </it>= 0.088, respectively). Strikingly, R0 patients with both high PAI-1 and uPAR-del4/5 mRNA expression levels showed a significant, 19-fold increased risk of tumor-related death (<it>P </it>= 0.044) compared to the low expression group.</p> <p>Conclusion</p> <p>Our results suggest that PAI-1 and uPAR-del4/5 mRNA levels may add prognostic information in STS patients with R0 status and distinguish a subgroup of R0 patients with low PAI-1 and/or low uPAR-del4/5 values who have a better outcome compared to patients with high marker levels.</p

Rational Diversification of a Promoter Providing Fine-Tuned Expression and Orthogonal Regulation for Synthetic Biology

Yeast is an ideal organism for the development and application of synthetic biology, yet there remain relatively few well-characterised biological parts suitable for precise engineering of this chassis. In order to address this current need, we present here a strategy that takes a single biological part, a promoter, and re-engineers it to produce a fine-graded output range promoter library and new regulated promoters desirable for orthogonal synthetic biology applications. A highly constitutive Saccharomyces cerevisiae promoter, PFY1p, was identified by bioinformatic approaches, characterised in vivo and diversified at its core sequence to create a 36-member promoter library. TetR regulation was introduced into PFY1p to create a synthetic inducible promoter (iPFY1p) that functions in an inverter device. Orthogonal and scalable regulation of synthetic promoters was then demonstrated for the first time using customisable Transcription Activator-Like Effectors (TALEs) modified and designed to act as orthogonal repressors for specific PFY1-based promoters. The ability to diversify a promoter at its core sequences and then independently target Transcription Activator-Like Orthogonal Repressors (TALORs) to virtually any of these sequences shows great promise toward the design and construction of future synthetic gene networks that encode complex “multi-wire” logic functions