The novel Fh8 and H fusion partners for soluble protein expression in Escherichia coli : a comparison with the traditional gene fusion technology

The Escherichia coli host system is an advantageous choice for simple and inexpensive recombinant protein production but it still presents bottlenecks at expressing soluble proteins from other organisms. Several efforts have been taken to overcome E. coli limitations, including the use of fusion partners that improve protein expression and solubility. New fusion technologies are emerging to complement the traditional solutions. This work evaluates two novel fusion partners, the Fh8 tag (8 kDa) and the H tag (1 kDa), as solubility enhancing tags in E. coli and their comparison to commonly used fusion partners. A broad range comparison was conducted in a small-scale screening and subsequently scaled-up. Six difficult-to-express target proteins (RVS167, SPO14, YPK1, YPK2, Frutalin and CP12) were fused to eight fusion tags (His, Trx, GST, MBP, NusA, SUMO, H and Fh8). The resulting protein expression and solubility levels were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis before and after protein purification and after tag removal. The Fh8 partner improved protein expression and solubility as the well-known Trx, NusA or MBP fusion partners. The H partner did not function as a solubility tag. Cleaved proteins from Fh8 fusions were soluble and obtained in similar or higher amounts than proteins from the cleavage of other partners as Trx, NusA or MBP. The Fh8 fusion tag therefore acts as an effective solubility enhancer, and its low molecular weight potentially gives it an advantage over larger solubility tags by offering a more reliable assessment of the target protein solubility when expressed as a fusion protein.The financial support of the EMBL Heidelberg, Germany and Fundacao para a Ciencia e Tecnologia (FCT), Portugal, is acknowledged: the fellowship SFRH/BD/46482/2008 to Sofia J. Costa and the project PTDC/CVT/103081/2008. The authors wish to acknowledge Anne-Claude Gavin for providing four of the constructs for this study (RVS167, SPO14, YPK1, and YPK2) and Emmanuel Poilpre for the experimental help (both from the EMBL Heidelberg, Germany)

Petrogenesis of rhyolite-trachyte-basalt composite ignimbrite P1, Gran Canaria, Canary Islands

The 14 Ma caldera-forming composite ignimbrite P1 on Gran Canaria (Canary Islands) represents the first voluminous eruption of highly differentiated magmas on top of the basaltic Miocene shield volcano. Compositional zonation of the ignimbrite is the result of vertically changing proportions of four component magmas, which were intensely mixed during eruption: (1) Crystal-poor to highly phyric rhyolite (∼10 km3), (2) sodic trachyandesite through mafic to evolved trachyte (∼6 km3), (3) Na-poor trachyandesite (<1 km3), and (4) basalt zoned from 5.2 to 4.3 wt % MgO (∼26 km3). P1 basalt is composed of two compositionally zoned magma batches, B2 basalt and B3 basalt. B3 basalt is derived from a mantle source depleted in incompatible trace elements compared to the shield basalt source. Basaltic magmas were stored in a reservoir probably underplating the crust, in which zoned B2 basaltic magma formed by mixing of “enriched” (shield) and “depleted” (B3) mafic melts and subsequent crystal fractionation. Evolved magmas formed in a shallow crustal chamber, whereas intermediate magmas formed at both levels. Abundant pyroxenitic to gabbroid cumulates in P1 support crystal fractionation as the major differentiation process. On the basis of major and trace element modeling, we infer two contemporaneous fractional crystallization series: series I from “enriched” shield basalt through Na-poor trachyandesite to rhyolite, and series II from “depleted” P1 basalt through sodic trachyandesite to trachyte. Series II rocks were significantly modified by selective contamination involving feldspar (Na, K, Ba, Eu, Sr), zircon (Zr) and apatite (P, Y, rare earth elements) components; apatite contamination also affected series I Na-poor trachyandesite. Substantial sodium introduction into sodic trachyandesite is the main reason for the different major element evolution of the two series, whereas their different parentage is mainly reflected in the high field strength trace elements. Selective element contamination involved not only rapidly but also slowly diffusing elements as well as different saturation conditions. Contamination processes thus variably involved differential diffusion, partial dissolution of minerals, partial melt migration, and trace mineral incorporation. Magma mixing between trachyte and rhyolite during their simultaneous crystallization in the P1 magma chamber is documented by mutual mineral inclusions but had little effect on the compositional evolution of both magmas. Fe-Ti oxide thermometry yields magmatic temperatures of around 850°C for crystal-poor through crystal-rich rhyolite, ∼815°C for trachyte and ∼850°–900°C for the trachyandesitic magmas. High 1160°C for the basalt magma suggest its intrusion into the P1 magma chamber only shortly before eruption. The lower temperature for trachyte compared to rhyolite and the strong crustal contamination of trachyte and sodic trachyandesite support their residence along the walls of the vertically and laterally zoned P1 magma chamber. The complex magmatic evolution of P1 reflects the transient state of Gran Canaria's mantle source composition and magma plumbing system during the change from basaltic to silicic volcanism. Our results for P1 characterize processes operating during this important transition, which also occurs on other volcanic ocean islands

Inborn errors of OAS-RNase L in SARS-CoV-2-related multisystem inflammatory syndrome in children

Multisystem inflammatory syndrome in children (MIS-C) is a rare and severe condition that follows benign COVID-19. We report autosomal recessive deficiencies of OAS1, OAS2, or RNASEL in five unrelated children with MIS-C. The cytosolic double-stranded RNA (dsRNA)-sensing OAS1 and OAS2 generate 2'-5'-linked oligoadenylates (2-5A) that activate the single-stranded RNA-degrading ribonuclease L (RNase L). Monocytic cell lines and primary myeloid cells with OAS1, OAS2, or RNase L deficiencies produce excessive amounts of inflammatory cytokines upon dsRNA or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) stimulation. Exogenous 2-5A suppresses cytokine production in OAS1-deficient but not RNase L-deficient cells. Cytokine production in RNase L-deficient cells is impaired by MDA5 or RIG-I deficiency and abolished by mitochondrial antiviral-signaling protein (MAVS) deficiency. Recessive OAS-RNase L deficiencies in these patients unleash the production of SARS-CoV-2-triggered, MAVS-mediated inflammatory cytokines by mononuclear phagocytes, thereby underlying MIS-C

CMS physics technical design report : Addendum on high density QCD with heavy ions

Peer reviewe

Ultrafast recombination for NiO sensitized with a series of perylene imide sensitizers exhibiting Marcus normal behaviour

Ultrafast recombination observed from several perylene imide sensitizers bound to NiO appears to align with Marcus normal region behaviour; this indicates recombination to intra-bandgap states

Cobalt Polypyridyl-Based Electrolytes for p-Type Dye-Sensitized Solar Cells

A series of polypyridyl cobalt complexes with different substituents was applied as redox mediators in p-type dye-sensitized solar cells (p-DSCs), consisting of mesoporous NiO sensitized with a perylenemonoimide naphthalenediimide (PMI-NDI) dyad. The photocurrent and photovoltages of the devices were found to depend on the steric bulk of the redox species rather than their electrochemical potential. Bulky substituents were found to slow the detrimental charge recombination reactions between holes in the NiO semiconductor and the reduced form of the redox couple. The open-circuit potential (V(OC)) of each of the devices was superior to the equivalent PMI-NDIsensitized p-DSCs containing the triiodide/iodide redox couple

Synthesis, photophysical and photovoltaic investigations of acceptor-functionalized perylene monoimide dyes for nickel oxide p-type dye-sensitized solar cells

We report on the synthesis, electrochem., photophys., and photovoltaic properties of a series of three org. dyads comprising a perylene monoimide (PMI) dye connected to a naphthalene diimide (NDI) or a fullerene (C60) for application in dye-sensitized solar cells (DSCs) with nanocryst. NiO electrodes. It was found that the secondary electron acceptor (NDI or C60) in all the three dyads extends the charge sepd. state lifetime by about five orders of magnitude compared to the resp. parent PMI dye. Nanosecond pump-probe expts. of the NiO/dyads in the presence of the electrolyte show that the redn. of triiodide by the secondary electron acceptor is slow in all the dyads, which we ascribe to a weak driving force for this reaction. This reaction is significantly faster with the cobalt electrolyte (tris(4,4'-di-tert-butyl-2,2'-bipyridine)cobalt(II/III)), whose driving force is larger; however, its reaction with the reduced dyads is still rather slow. We demonstrate that the larger photovoltage obsd. with the cobalt electrolyte (VOC = 285 mV) relative to the iodide electrolyte (VOC = 120 mV) is due to a decrease in the dark current for the former owing to slower interfacial electron transfer of the reduced mediator with the injected holes into the NiO electrode. In terms of photovoltaic performances, the most efficient dyad is the system in which the NDI is directly connected to the PMI (η = 0.14% under AM 1.5 with the cobalt electrolyte), but the dyad contg. the fullerene acceptor exhibits the highest IPCE and the highest short circuit c.d. (IPCE = 57%, JSC = 1.88 mA cm-2) with the iodide electrolyte. The latter performances are attributed to the slightly stronger reducing power of C60 relative to NDI, which favors the redn. of the mediator in the electrolyte