Prolonged activity of the pestiviral RNase Erns as an interferon antagonist after uptake by clathrin-mediated endocytosis

The RNase activity of the envelope glycoprotein E(rns) of the pestivirus bovine viral diarrhea virus (BVDV) is required to block type I interferon (IFN) synthesis induced by single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA) in bovine cells. Due to the presence of an unusual membrane anchor at its C terminus, a significant portion of E(rns) is also secreted. In addition, a binding site for cell surface glycosaminoglycans is located within the C-terminal region of E(rns). Here, we show that the activity of soluble E(rns) as an IFN antagonist is not restricted to bovine cells. Extracellularly applied E(rns) protein bound to cell surface glycosaminoglycans and was internalized into the cells within 1 h of incubation by an energy-dependent mechanism that could be blocked by inhibitors of clathrin-dependent endocytosis. E(rns) mutants that lacked the C-terminal membrane anchor retained RNase activity but lost most of their intracellular activity as an IFN antagonist. Surprisingly, once taken up into the cells, E(rns) remained active and blocked dsRNA-induced IFN synthesis for several days. Thus, we propose that E(rns) acts as an enzymatically active decoy receptor that degrades extracellularly added viral RNA mainly in endolysosomal compartments that might otherwise activate intracellular pattern recognition receptors (PRRs) in order to maintain a state of innate immunotolerance. IMPORTANCE The pestiviral RNase E(rns) was previously shown to inhibit viral ssRNA- and dsRNA-induced interferon (IFN) synthesis. However, the localization of E(rns) at or inside the cells, its species specificity, and its mechanism of interaction with cell membranes in order to block the host's innate immune response are still largely unknown. Here, we provide strong evidence that the pestiviral RNase E(rns) is taken up within minutes by clathrin-mediated endocytosis and that this uptake is mostly dependent on the glycosaminoglycan binding site located within the C-terminal end of the protein. Remarkably, the inhibitory activity of E(rns) remains for several days, indicating the very potent and prolonged effect of a viral IFN antagonist. This novel mechanism of an enzymatically active decoy receptor that degrades a major viral pathogen-associated molecular pattern (PAMP) might be required to efficiently maintain innate and, thus, also adaptive immunotolerance, and it might well be relevant beyond the bovine species.

Syncytiotrophoblast vesicles show altered micro-RNA and haemoglobin content after ex-vivo perfusion of placentas with haemoglobin to mimic preeclampsia.

BACKGROUND Cell-free foetal haemoglobin (HbF) has been shown to play a role in the pathology of preeclampsia (PE). In the present study, we aimed to further characterize the harmful effects of extracellular free haemoglobin (Hb) on the placenta. In particular, we investigated whether cell-free Hb affects the release of placental syncytiotrophoblast vesicles (STBMs) and their micro-RNA content. METHODS The dual ex-vivo perfusion system was used to perfuse isolated cotyledons from human placenta, with medium alone (control) or supplemented with cell-free Hb. Perfusion medium from the maternal side of the placenta was collected at the end of all perfusion phases. The STBMs were isolated using ultra-centrifugation, at 10,000×g and 150,000×g (referred to as 10K and 150K STBMs). The STBMs were characterized using the nanoparticle tracking analysis, identification of surface markers and transmission electron microscopy. RNA was extracted and nine different micro-RNAs, related to hypoxia, PE and Hb synthesis, were selected for analysis by quantitative PCR. RESULTS All micro-RNAs investigated were present in the STBMs. Mir-517a, mir-141 and mir-517b were down regulated after Hb perfusion in the 10K STBMs. Furthermore, Hb was shown to be carried by the STBMs. CONCLUSION This study showed that Hb perfusion can alter the micro-RNA content of released STBMs. Of particular interest is the alteration of two placenta specific micro-RNAs; mir-517a and mir-517b. We have also seen that STBMs may function as carriers of Hb into the maternal circulation.

Efficacy and safety of a novel bioabsorbable polymer-coated, everolimus-eluting coronary stent: the EVOLVE II Randomized Trial.

BACKGROUND Drug eluting stents with durable polymers may be associated with hypersensitivity, delayed healing, and incomplete endothelialization, which may contribute to late/very late stent thrombosis and the need for prolonged dual antiplatelet therapy. Bioabsorbable polymers may facilitate stent healing, thus enhancing clinical safety. The SYNERGY stent is a thin-strut, platinum chromium metal alloy platform with an ultrathin bioabsorbable Poly(D,L-lactide-co-glycolide) abluminal everolimus-eluting polymer. We performed a multicenter, randomized controlled trial for regulatory approval to determine noninferiority of the SYNERGY stent to the durable polymer PROMUS Element Plus everolimus-eluting stent. METHODS AND RESULTS Patients (n=1684) scheduled to undergo percutaneous coronary intervention for non-ST-segment-elevation acute coronary syndrome or stable coronary artery disease were randomized to receive either the SYNERGY stent or the PROMUS Element Plus stent. The primary end point of 12-month target lesion failure was observed in 6.7% of SYNERGY and 6.5% PROMUS Element Plus treated subjects by intention-to-treat (P=0.83 for difference; P=0.0005 for noninferiority), and 6.4% in both the groups by per-protocol analysis (P=0.0003 for noninferiority). Clinically indicated revascularization of the target lesion or definite/probable stent thrombosis were observed in 2.6% versus 1.7% (P=0.21) and 0.4% versus 0.6% (P=0.50) of SYNERGY versus PROMUS Element Plus-treated subjects, respectively. CONCLUSIONS In this randomized trial, the SYNERGY bioabsorbable polymer everolimus-eluting stent was noninferior to the PROMUS Element Plus everolimus-eluting stent with respect to 1-year target lesion failure. These data support the relative safety and efficacy of SYNERGY in a broad range of patients undergoing percutaneous coronary intervention. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT01665053.

The Impact of Thermal Coupling on Catalyst Behavior in Wall-Coated Heat-Exchanger Microreactors

This thesis presents a numerical study of reaction and diffusion phenomena in wall-coated heat-exchanger microreactors. Specifically, the interactions between an endothermic and exothermic catalyst layer separated by an impermeable wall is studied to understand the inherent behavior of the system. Two modeling approaches are presented, the first under the assumption of a constant thermal gradient and neglecting heat of reaction and the second considering both catalyst layers and reaction heat. Both studies found that thicker, more thermally insulating catalyst layers increase the effectiveness of the exothermic reaction by allowing for accumulation of reaction heat while thinner catalyst layers for the endothermic catalyst allow for direct access of the reactant to higher wall temperatures.

Influence of mycobacterial trehalose 6,6'-dimycolate (TDM) on macrophage responses

Mycobacterium tuberculosis, the causative agent of tuberculosis, survives within macrophages by altering host cell activation and by manipulating phagosomal trafficking and acidification. Part of the success of M. tuberculosis as a major human pathogen has been attributed to its cell wall, a unique structure largely comprised of mycolic acids. Trehalose 6,6′-dimycolate (TDM) is the major glycolipid component on the surface of the mycobacterial cell wall. This study examines the contribution of TDM during mycobacterial infection of murine macrophages. Virulent M. tuberculosis was chemically depleted of surface-exposed TDM using petroleum ether extraction. Compared to their native counterparts, delipidated M. tuberculosis showed similar growth in broth culture. Bone marrow-derived macrophages (BMM) or the murine macrophage-like cell line J774A.1 were infected with delipidated M. tuberculosis, and responses were compared to cells infected with native M. tuberculosis. Delipidated M. tuberculosis demonstrated significantly decreased viability in macrophages by seven days after infection. Reconstitution of delipidated organisms with pure TDM restored viability. Infection with native M. tuberculosis led to high cellular production of cytokines (IL-1β, IL-6, IL-12, and TNF-α) and chemokines (MCP-1 and MIP-1α); infection with delipidated M. tuberculosis significantly abrogated responses. Cytokine and chemokine production were restored when delipidated organisms were reconstituted with TDM. Responses were specifically induced by TDM; all measured cytokines were elicited from macrophages incubated with TDM-coated beads, while control beads coated with bovine serum albumin (BSA) did not induce cytokine production. Visualization of mycobacterial localization in J774A.1 cells using fluorescence microscopy revealed that delipidated M. tuberculosis were significantly more likely to traffic to acidic vesicles (lysosomes) than native organisms. Reconstitution with TDM restored trafficking to non-acidic vesicles. Similarly, TDM-coated beads demonstrated significantly delayed localization to acidic vesicles compared to BSA-coated beads. In summary, the interaction of TDM with macrophages may regulate the outcome of M. tuberculosis infection by influencing cellular cytokine production and intracellular localization of organisms. This research has elucidated a novel and necessary role for TDM in survival of virulent M. tuberculosis in host macrophages during in vitro infection. ^

Microfossils detected in a block of ancient dense clay coated with a ferromanganese crust, Clarion-Clipperton Province, East Equatorial Pacific

The area in study is characterized by a regional stratigraphic hiatus from Early Miocene to Quaternary. Deposits from Late Eocene to Early Miocene occur on the bottom surface or under a thin sedimentary cover. Ferromanganese nodules, mostly of Oligocene age, formed on surface layers of Tertiary or Quaternary sediments. A detailed micropaleontological study of a block of dense ancient clay coated with a ferromanganese crust was carried out. Composition of found radiolarian and diatomaceous complexes proved that the crust formed in Quaternary on an eroded surface of Late Oligocene clay. In Quaternary Neogene sediments were eroded and washed away by bottom currents. It is likely that the erosion began 0.9-0.7 Ma at the beginning of the "Glacial Pleistocene". The erosion could be initiated by loosening and resuspension of surface sediments resulting from seismic activity generated by strong earthquakes in the Central America subduction zone. The same vibration maintained residual nodules at the seafloor surface. Thus, for the area in study a common reason and a common Quaternary interval for formation of the following features is supposed: a regional stratigraphic hiatus, formation of residual nodule fields, and position of ancient nodules on the surface of Quaternary sediments.

Composition of bioclastic sands, carbonates and pyroclastic rocks of the Great Meteor and Josephine Seamounts, eastern North Atlantic

1. Great Meteor Seamount (GMS) is a very large (24,000 km**3) guyot with a flat summit plateau at 330-275 m; it has a volcanic core, capped by 150-600 m of post-Middle-Miocene carbonate and pyroclastic rocks, and is covered by bioclastic sands. The much smaller Josephine Seamount (JS, summit 170- 500 m w. d.) consists mainly of basalt which is only locally covered by limestones and bioclastic sands. 2. The bioclastic sands are almost free of terrigenous components, and are well sorted, unimodal medium sands. (1) "Recent pelagic sands" are typical of water depths > 600 m (JS) or > 1000 m (GMS). (2) "Sands of mixed relict-recent origin" (10-40% relict) and (3) "relict sands" (> 40% relict) are highly reworked, coarse lag deposits from the upper flanks and summit tops in which recent constituents are mixed with Pleistocene or older relict material. 3. From the carbonate rocks of both seamounts, 12 "microfacies" (MF-)types were distinguished. The 4 major types are: (1) Bio(pel)sparites (MF 1) occur on the summit plateaus and consist of magnesian calcite cementing small pellets and either redeposited planktonic bioclasts or mixed benthonic-planktonic skeletal debris ; (2) Porous biomicrites (MF 2) are typical of the marginal parts of the summit plateaus and contain mostly planktonic foraminifera (and pteropods), sometimes with redeposited bioclasts and/or coated grains; (3) Dense, ferruginous coralline-algal biomicrudites with Amphistegina sp. (MF 3.1), or with tuffaceous components (MF 3.2); (4) Dense, pelagic foraminiferal nannomicrite (MF 4) with scattered siderite rhombs. Corresponding to the proportion and mineralogical composition of the bioclasts and of the (Mgcalcitic) peloids, micrite, and cement, magnesian calcite (13-17 mol-% MgCO3) is much more abundant than low-Mg calcite and aragonite in rock types (1) and (2). Type (3) contains an "intermediate" Mg-calcite (7-9 mol-X), possibly due to an original Mg deficiency or to partial exsolution of Mg during diagenesis. The nannomicrite (4) consists of low-Mg calcite only. 4. Three textural types of volcanic and associated gyroclastic rocks were distinguished: (1) holohyaline, rapidly chilled and granulated lava flows and tuffs (palagonite tuff breccia and hyaloclastic top breccia); (2) tachylitic basalts (less rapidly chilled; with opaque glass); and (3) "slowly" crystallized, holocrystalline alkali olivine basalts. The carbonate in most mixed pyroclastic-carbonate sediments at the basalt contact is of "post-eruptive" origin (micritic crusts etc.); "pre-eruptive" limestone is recrystallized or altered at the basalt contact. A deuteric (?hydrothermal) "mineralX", filling vesicles in basalt and cementing pyroclastic breccias is described for the first time. 5. Origin and development of GMS andJS: From its origin, some 85 m. y. ago, the volcano of GMS remained active until about 10 m. y. B. P. with an average lava discharge of 320 km**3/m. y. The volcanic origin of JS is much younger (?Middle Tertiary), but the volcanic activity ended also about 9 m. y. ago. During L a t e Miocene to Pliocene times both volcanoes were eroded (wave-rounded cobbles). The oldest pyroclastics and carbonates (MF 3.1, 3.2) were originally deposited in shallow-water (?algal reef hardground). The Plio (-Pleisto) cene foraminiferal nannomicrites (MF 4) suggest a meso- to bathypelagic environment along the flanks of GMS. During the Quaternary (?Pleistocene) bioclastic sands were deposited in water depths beyond wave base on the summit tops, repeatedly reworked, and lithified into loosely consolidated biopelsparites and biomicrites (MF 1 and 2; Fig. 15). Intermediate steps were a first intragranular filling by micrite, reworking, oncoidal coating, weak consolidation with Mg-calcite cemented "peloids" in intergranular voids and local compaction of the peloids into cryptocrystalline micrite with interlocking Mg-calcite crystals up to 4p. The submarine lithification process was frequently interrupted by long intervals of nondeposition, dissolution, boring, and later infilling. The limestones were probably never subaerially exposed. Presently, the carbonate rocks undergo biogenic incrustation and partial dissolution into bioclastic sands. The irregular distribution pattern of the sands reflects (a) the patchy distribution of living benthonic organisms, (b) the steady rain of planktonic organism onto the seamount top, (c) the composition of disintegrating subrecent limestones, and (d) the intensity of winnowing and reworking bottom current

(Table 1) Minerals found in vesicles and fractures from DSDP Leg 54 Holes basalts

A variety of secondary minerals, formed in response to different oxidation and hydration states, are found in vugs and on fracture surfaces of the basalt cores from DSDP Leg 54. The minerals are smectite (blue to grey), high-magnesium calcite, manganoan calcite, aragonite, iron oxides, phillipsite, todorokite, marcasite, and hydrobiotite. The relationship of the mineral assemblages to four depositional modes of the basalts are delineated. A definite sequence and genetic link exists between mineral type and host rock which is dependent upon the origin and subsequent cooling history of the basalt.