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

Autoantibodies neutralizing type I IFNs are present in ~4% of uninfected individuals over 70 years old and account for ~20% of COVID-19 deaths

Circulating autoantibodies (auto-Abs) neutralizing high concentrations (10 ng/mL, in plasma diluted 1 to 10) of IFN-α and/or -ω are found in about 10% of patients with critical COVID-19 pneumonia, but not in subjects with asymptomatic infections. We detect auto-Abs neutralizing 100-fold lower, more physiological, concentrations of IFN-α and/or -ω (100 pg/mL, in 1/10 dilutions of plasma) in 13.6% of 3,595 patients with critical COVID-19, including 21% of 374 patients &gt; 80 years, and 6.5% of 522 patients with severe COVID-19. These antibodies are also detected in 18% of the 1,124 deceased patients (aged 20 days-99 years; mean: 70 years). Moreover, another 1.3% of patients with critical COVID-19 and 0.9% of the deceased patients have auto-Abs neutralizing high concentrations of IFN-β. We also show, in a sample of 34,159 uninfected subjects from the general population, that auto-Abs neutralizing high concentrations of IFN-α and/or -ω are present in 0.18% of individuals between 18 and 69 years, 1.1% between 70 and 79 years, and 3.4% &gt;80 years. Moreover, the proportion of subjects carrying auto-Abs neutralizing lower concentrations is greater in a subsample of 10,778 uninfected individuals: 1% of individuals &lt;70 years, 2.3% between 70 and 80 years, and 6.3% &gt;80 years. By contrast, auto-Abs neutralizing IFN-β do not become more frequent with age. Auto-Abs neutralizing type I IFNs predate SARS-CoV-2 infection and sharply increase in prevalence after the age of 70 years. They account for about 20% of both critical COVID-19 cases in the over-80s, and total fatal COVID-19 cases

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

Rare predicted loss-of-function variants of type I IFN immunity genes are associated with life-threatening COVID-19

Background: We previously reported that impaired type I IFN activity, due to inborn errors of TLR3- and TLR7-dependent type I interferon (IFN) immunity or to autoantibodies against type I IFN, account for 15–20% of cases of life-threatening COVID-19 in unvaccinated patients. Therefore, the determinants of life-threatening COVID-19 remain to be identified in ~ 80% of cases. Methods: We report here a genome-wide rare variant burden association analysis in 3269 unvaccinated patients with life-threatening COVID-19, and 1373 unvaccinated SARS-CoV-2-infected individuals without pneumonia. Among the 928 patients tested for autoantibodies against type I IFN, a quarter (234) were positive and were excluded. Results: No gene reached genome-wide significance. Under a recessive model, the most significant gene with at-risk variants was TLR7, with an OR of 27.68 (95%CI 1.5–528.7, P = 1.1 × 10−4) for biochemically loss-of-function (bLOF) variants. We replicated the enrichment in rare predicted LOF (pLOF) variants at 13 influenza susceptibility loci involved in TLR3-dependent type I IFN immunity (OR = 3.70[95%CI 1.3–8.2], P = 2.1 × 10−4). This enrichment was further strengthened by (1) adding the recently reported TYK2 and TLR7 COVID-19 loci, particularly under a recessive model (OR = 19.65[95%CI 2.1–2635.4], P = 3.4 × 10−3), and (2) considering as pLOF branchpoint variants with potentially strong impacts on splicing among the 15 loci (OR = 4.40[9%CI 2.3–8.4], P = 7.7 × 10−8). Finally, the patients with pLOF/bLOF variants at these 15 loci were significantly younger (mean age [SD] = 43.3 [20.3] years) than the other patients (56.0 [17.3] years; P = 1.68 × 10−5). Conclusions: Rare variants of TLR3- and TLR7-dependent type I IFN immunity genes can underlie life-threatening COVID-19, particularly with recessive inheritance, in patients under 60&nbsp;years old

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