Structural basis for CRISPR RNA-guided DNA recognition by Cascade

The CRISPR (clustered regularly interspaced short palindromic repeats) immune system in prokaryotes uses small guide RNAs to neutralize invading viruses and plasmids. In Escherichia coli, immunity depends on a ribonucleoprotein complex called Cascade. Here we present the composition and low-resolution structure of Cascade and show how it recognizes double-stranded DNA (dsDNA) targets in a sequence-specific manner. Cascade is a 405-kDa complex comprising five functionally essential CRISPR-associated (Cas) proteins (CasA1B2C6D1E1) and a 61-nucleotide CRISPR RNA (crRNA) with 5′-hydroxyl and 2′,3′-cyclic phosphate termini. The crRNA guides Cascade to dsDNA target sequences by forming base pairs with the complementary DNA strand while displacing the noncomplementary strand to form an R-loop. Cascade recognizes target DNA without consuming ATP, which suggests that continuous invader DNA surveillance takes place without energy investment. The structure of Cascade shows an unusual seahorse shape that undergoes conformational changes when it binds target DNA.

Delivery of sTRAIL variants by MSCs in combination with cytotoxic drug treatment leads to p53-independent enhanced antitumor effects

Mesenchymal stem cells (MSCs) are able to infiltrate tumor tissues and thereby effectively deliver gene therapeutic payloads. Here, we engineered murine MSCs (mMSCs) to express a secreted form of the TNF-related apoptosis-inducing ligand (TRAIL), which is a potent inducer of apoptosis in tumor cells, and tested these MSCs, termed MSC.sTRAIL, in combination with conventional chemotherapeutic drug treatment in colon cancer models. When we pretreated human colorectal cancer HCT116 cells with low doses of 5-fluorouracil (5-FU) and added MSC.sTRAIL, we found significantly increased apoptosis as compared with single-agent treatment. Moreover, HCT116 xenografts, which were cotreated with 5-FU and systemically delivered MSC.sTRAIL, went into remission. Noteworthy, this effect was protein 53 (p53) independent and was mediated by TRAIL-receptor 2 (TRAIL-R2) upregulation, demonstrating the applicability of this approach in p53-defective tumors. Consequently, when we generated MSCs that secreted TRAIL-R2-specific variants of soluble TRAIL (sTRAIL), we found that such engineered MSCs, labeled MSC.sTRAIL DR5, had enhanced antitumor activity in combination with 5-FU when compared with MSC.sTRAIL. In contrast, TRAIL-resistant pancreatic carcinoma PancTu1 cells responded better to MSC.sTRAIL DR4 when the antiapoptotic protein XIAP (X-linked inhibitor of apoptosis protein) was silenced concomitantly. Taken together, our results demonstrate that TRAIL-receptor selective variants can potentially enhance the therapeutic efficacy of MSC-delivered TRAIL as part of individualized and tumor-specific combination treatments. © 2013 Macmillan Publishers Limited All rights reserved

Impact of liquid metal surface on plasma-surface interaction in experiments with lithium and tin capillary porous systems

The lithium and tin capillary-porous systems (CPSs) were tested with steady-state plasma in the PLM plasma device which is the divertor simulator with plasma parameters relevant to divertor and SOL plasma of tokamaks. The CPS consists of tin/lithium tile fixed between two molybdenum meshs constructed in the module faced to plasma. Steady-state plasma load of 0.1 - 1 MW/m(2) on the CPS during more than 200 min was achieved in experiments on PLM which is a modeling far scrapeoff- layer and far zone of divertor plasma of a large tokamak. The heating of the CPS was controlled remotely including biasing technique which allows to regulate evaporated metal influx to plasma. After exposure, the materials of the tin and lithium CPSs were inspected and analyzed with optic and scanning electron micriscopy. Experiments have demonstrated sustainability of the tin and lithium CPSs to the high heat steady state plasma load expected in a large scale tokamak. The effect of evaporated lithium and tin on the plasma transport/radiation was studied with spectroscopy to evaluate changes of plasma properties and plasma-surface interaction

Complex Risks from Old Urban Waste Landfills: Sustainability Perspective from Iasi, Romania

Landfills continue to represent the most frequent managerial practice for municipal solid wastes and an increasing and complex problem globally. In certain countries, a transition to an open society and free market is superimposed on the transition to sustainability, resulting in even higher complexity of management. This paper proposes an approach for problem-structuring of landfills in complex transitions: sustainability or unsustainability of a management approach is determined by a set of sustainability filters that are defined by sets of indicators and prioritized according the systemic concept of sustainability, which says that economy is embedded in society, which is embedded in nature. The writers exercise this approach with an old landfill in Iasi, Romania, and conclude for unsustainability, because the ecological sustainability filter is not successfully passed. Social and economic sustainability filters are also discussed in relation with the ecological sustainability indicators. The described approach allows a coherent, transdisciplinary synthesis of knowledge scattered across various disciplines, a pervasive problem in landfill management. The case study helps distinguish between generally true and context-dependent aspects.Peer reviewe

Laboratory evolution of Pyrococcus furiosus alcohol dehydrogenase to improve the production of (2S,5S)-hexanediol at moderate temperatures

There is considerable interest in the use of enantioselective alcohol dehydrogenases for the production of enantio- and diastereomerically pure diols, which are important building blocks for pharmaceuticals, agrochemicals and fine chemicals. Due to the need for a stable alcohol dehydrogenase with activity at low-temperature process conditions (30°C) for the production of (2S,5S)-hexanediol, we have improved an alcohol dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus (AdhA). A stable S-selective alcohol dehydrogenase with increased activity at 30°C on the substrate 2,5-hexanedione was generated by laboratory evolution on the thermostable alcohol dehydrogenase AdhA. One round of error-prone PCR and screening of ∼1,500 mutants was performed. The maximum specific activity of the best performing mutant with 2,5-hexanedione at 30°C was tenfold higher compared to the activity of the wild-type enzyme. A 3D-model of AdhA revealed that this mutant has one mutation in the well-conserved NADP(H)-binding site (R11L), and a second mutation (A180V) near the catalytic and highly conserved threonine at position 183

Thermal Stabilization of an Endoglucanase by Cyclization

An intein-driven protein splicing approach allowed for the covalent linkage between the N- and C-termini of a polypeptide chain to create circular variants of the endo-β-1,3-1,4-glucanase, LicA, from Bacillus licheniformis. Two circular variants, LicA-C1 and LicA-C2, which have connecting loops of 20 and 14 amino acids, respectively, showed catalytic activities that are approximately two and three times higher, respectively, compared to that of the linear LicA (LicA-L1). The thermal stability of the circular variants was significantly increased compared to the linear form. Whereas the linear glucanase lost half of its activity after 3 min at 65 °C, the two circular variants have 6-fold (LicA-C1) and 16-fold (LicA-C2) increased half-life time of inactivation. In agreement with this, fluorescence spectroscopy and differential scanning calorimetry studies revealed that circular enzymes undergo structural changes at higher temperatures compared to that of the linear form. The effect of calcium on the conformational stability and function of the circular LicAs was also investigated, and we observed that the presence of calcium ions results in increased thermal stability. The impact of the length of the designed loops on thermal stability of the circular proteins is discussed, and it is suggested that cyclization may be an efficient strategy for the increased stability of proteins

Low N2_{2}O and variable CH4_{4} fluxes from tropical forest soils of the Congo Basin

Globally, tropical forests are assumed to be an important source of atmospheric nitrous oxide (N$_{2}$O) and sink for methane (CH$_{4}$). Yet, although the Congo Basin comprises the second largest tropical forest and is considered the most pristine large basin left on Earth, in situ N$_{2}$O and CH$_{4}$ flux measurements are scarce. Here, we provide multi-year data derived from on-ground soil flux (n = 1558) and riverine dissolved gas concentration (n = 332) measurements spanning montane, swamp, and lowland forests. Each forest type core monitoring site was sampled at least for one hydrological year between 2016 - 2020 at a frequency of 7-14 days. We estimate a terrestrial CH$_{4}$ uptake (in kg CH$_{4}$-C ha$^{-1}$ yr$^{-1}$) for montane (−4.28) and lowland forests (−3.52) and a massive CH$_{4}$ release from swamp forests (non-inundated 2.68; inundated 341). All investigated forest types were a N$_{2}$O source (except for inundated swamp forest) with 0.93, 1.56, 3.5, and −0.19 kg N$_{2}$O-N ha$^{-1}$ yr$^{-1}$ for montane, lowland, non-inundated swamp, and inundated swamp forests, respectively