New insights on the role of paired membrane structures in coronavirus replication.

The replication of coronaviruses, as in other positive-strand RNA viruses, is closely tied to the formation of membrane-bound replicative organelles inside infected cells. The proteins responsible for rearranging cellular membranes to form the organelles are conserved not just among the Coronaviridae family members, but across the order Nidovirales. Taken together, these observations suggest that the coronavirus replicative organelle plays an important role in viral replication, perhaps facilitating the production or protection of viral RNA. However, the exact nature of this role, and the specific contexts under which it is important have not been fully elucidated. Here, we collect and interpret the recent experimental evidence about the role and importance of membrane-bound organelles in coronavirus replication.

Vasculogenesis: A process of vessel formation and its role during Ewing's sarcoma development

Vasculogenesis is the process by which Endothelial Precursor Cells (EPCs) form a vasculature. This process has been traditionally regarded as an embryological process of vessel formation. However, as early as in the 60's the concept of postnatal vasculogenesis was introduced, with a strong resurface of this idea in recent years. Similarly, previous work on a mouse skin tumor model provided us with the grounds to consider the role of vasculogenesis during tumor formation. ^ We examined the contribution of donor bone marrow (BM)-derived cells to neovascularization in recipient nude mice with Ewing's sarcoma. Ewing's sarcoma is a primitive neuroectodermal tumor that most often affects children and young adults between 5 and 30 years of age. Despite multiple attempts to improve the efficacy of chemotherapy for the disease, the 2-year metastases-free survival rate for patients with Ewing's sarcoma has not improved over the past 15 years. New therapeutic approaches are therefore needed to reduce the mortality rate. ^ The contribution of BM endothelial precursor cells in the development of Ewing's sarcoma was examined using different strategies to track the donor-derived cells. Using a BMT model that takes advantage of MHC differences between donor and recipient mice, we have found that donor BM cells were involved in the formation of Ewing's sarcoma vasculature. ^ Cells responsible for this vasculogenesis activity may be located within the stem cell population of the murine BM. These stem cells would not only generate the hematopoietic lineage but they would also generate ECs. Bone marrow SP (Side Population) cells pertain to a subpopulation that can be identified using flow cytometric analysis of Hoechst 33342-stained BM. This population of cells has HSC activity. We have tested the ability of BM SP cells to contribute to vasculogenesis in Ewing's sarcoma using our MHC mismatched transplant model. Mice transplanted with SP cells developed tumor neovessels that were derived from the donor SP cells. Thus, SP cells not only replenished the hematopoietic system of the lethally irradiated mice, but also differentiated into a non-hematopoietic cell lineage and contributed to the formation of the tumor vasculature. ^ In summary, we have demonstrated that BM-derived cells are involved in the generation of the new vasculature during the growth of Ewing's sarcoma. The finding that vasculogenesis plays a role in Ewing's sarcoma development opens the possibility of using genetically modified BM-derived cells for the treatment of Ewing's sarcomas. ^

The contributions of the amygdala, orbital frontal cortex and hippocampal formation to primate social cognition

Deficits in social cognition are prominent symptoms of many human psychiatric disorders, but the origin of such deficits remains largely unknown. To further current knowledge regarding the neural network mediating social cognition, the present research program investigated the individual contributions of two temporal lobe structures, the amygdala and hippocampal formation, and one frontal lobe region, the orbital frontal cortex (Areas 11 and 13), to primate social cognition. Based on previous research, we hypothesized that the amygdala, hippocampal formation and orbital frontal cortex contribute significantly to the formation of new social relationships, but less to the maintenance of familiar ones. ^ Thirty-six male rhesus macaques (Macaca mulatta) served as subjects, and were divided into four experimental groups: Neurotoxic amygdala lesion (A-ibo, n = 9), neurotoxic or aspiration orbital frontal cortex lesion (O, n = 9), neurotoxic hippocampal formation lesion (H-ibo, n = 9) or sham-operated control (C, n = 9). Six social groups (tetrads) were created, each containing one member from each experimental group. The effect of lesion on established social relationships was assessed during pre- and post-surgical unrestrained social interactions, whereas the effect of lesion on the formation of new relationships was assessed during an additional phase of post-surgical testing with shuffled tetrad membership. Results indicated that these three neural structures each contribute significantly to both the formation and maintenance of social relationships. Furthermore, the amygdala appears to primarily mediate normal responses to threatening social signals, whereas the orbital frontal cortex plays a more global role in social cognition by mediating responses to both threatening and affiliative social signals. By contrast, the hippocampal formation seems to contribute to social cognition indirectly by providing access to previous experience during social judgments. ^ These conclusions were further investigated with three experiments that measured behavioral and physiological (stress hormone) reactivity to threatening stimuli, and three additional experiments that measured subjects' ability to flexibly alter behavioral responses depending on the incentive value of a food reinforcer. Data from these six experiments further confirmed and strengthened the three conclusions originating from the social behavior experiments and, when combined with the current literature, helped to formulate a simple, but testable, theoretical model of primate social cognition. ^

Identification and characterization of new genes involved in Drosophila heart development

Heart development is a crucial and conserved process that is related to the major type of human birth defects. Dorsal vessel, the Drosophila heart, has been regarded as an insightful system to identify new genes and study gene functions involved in heart development. Using heart-specific GFP transgenes, I did a genetic screen for cardiogenic genes on Drosophila chromosome II. Drosophila mutants that carry chromosome II deficiencies were tested for their phenotypes of heart development. Based on the screen results, chromosome regions containing genes required for heart development were identified. Fly strains with single gene mutations located within the defined deficiency regions were tested further. Seven genes have been identified to be involved in heart development. ^ The LIM homeodomain transcription factor gene tailup (tup) was further studied for its function in heart development. Based on this study, tup is expressed in cardioblasts and pericardial cells of the heart tube, as well as in associated lymph glands and alary muscles. In depth analysis of tup mutant phenotypes demonstrated tup is required for normal development of both heart and lymph glands. Tup was shown to bind to two DNA recognition sequences in the dorsal vessel enhancer of the Hand bHLH transcription factor gene, with one site proven essential for the expression of Hand in lymph glands, pericardial cells, and Svp/Doc cardioblasts. Together, these studies demonstrate that Tup is a critical new transcription factor in dorsal vessel morphogenesis and lymph gland formation, and strongly suggest Tup is a direct regulator of the expression of Hand in these developmental processes. ^

A new fork for clinical application: Targeting forkhead transcription factors in cancer therapy

RAS-ERK-MAPK (Mitogen-activated protein kinase) pathway plays an essential role in proliferation, differentiation, and tumor progression. In this study, we showed that ERK downregulated FOXO3a through directly interacting with and phosphorylating FOXO3a at Serine 294, Serine 344, and Serine 425. ERK-phosphorylated FOXO3a was degraded by MDM2-mediated ubiquitin-proteosome pathway. FOXO3a phosphorylation and degradation consequently promoted cell proliferation and tumorigenesis. However, the non-phosphorylated FOXO3a mutant, which was resistant to the interaction and degradation by MDM2, resulted in inhibition of tumor formation. Forkhead O transcription factors (FOXOs) are important in the regulation of cellular functions including cell cycle arrest and cell death. Perturbation of FOXOs function leads to deregulated cell proliferation and cancer. Inactivation of FOXO proteins by activation of cell survival pathways, such as PI3K/AKT/IKK, is associated with tumorigenesis. Our study will further highlight FOXOs as new therapeutic targets in a broad spectrum of cancers. ^ Chemotherapeutic drug resistance is the most concerned problem in cancer therapy as resistance ultimately leads to treatment failure of cancer patients. In another study, we showed that blocking ERK activity with AZD6244, an established MEK1/2 inhibitor currently under human cancer clinical trials, enhances FOXO3a expression in various human cancer cell lines in vitro, and also in human colon cancer cell xenografts in vivo. Knocking down FOXO3a and its downstream gene Bim impaired AZD6244-induced growth suppression, whereas restoring activation of FOXO3a sensitized human cancer cell to AZD6244-induced growth arrest and apoptosis. More importantly, AZD6244-resistant cancer cells showed impaired endogenous FOXO3a nuclear translocation, reduced FOXO3a-Bim promoter association and significantly decreased Bim expression in response to AZD6244. AZD6244-resistant cancer cells can be sensitized to API-2 (an AKT inhibitor) and LY294002 (a PI3K inhibitor) in suppressing cell growth and colony formation, these inhibitors were known to enhance FOXO3a activity/nuclear translocation through inhibiting PI3K-AKT pathway. This study reveals novel molecular mechanism contributing to AZD6244-resistance through regulation of FOXO3a activity, further provides significant clinical implication of combining AZD6244 with PI3K/AKT inhibitors for sensitizing AZD6244-resistant cancer cells by activating FOXO3a. FOXO3a activation can be an essential pharmacological target and indicator to mediate and predict AZD6244 efficacy in clinical use. ^

Spatial compartmentalization of free radical formation and mitochondrial heterogeneity in bivalve gills revealed by live-imaging techniques, supplementary material

Live-imaging techniques (LIT) utilize target-specific fluorescent dyes to visualize biochemical processes using confocal and multiphoton scanning microscopy, which are increasingly employed as non-invasive approach to physiological in-vivo and ex-vivo studies. Here we report application of LIT to bivalve gills for ex-vivo analysis of gill physiology and mapping of reactive oxygen (ROS) and nitrogen (RNS) species formation in the living tissue. Our results indicate that H2O2, HOO. and ONOO- radicals (assessed through C-H2DFFDA staining) are mainly formed within the blood sinus of the filaments and are likely to be produced by hemocytes as defense against invading pathogens. The oxidative damage in these areas is controlled by enhanced CAT (catalase) activities recorded within the filaments. The outermost areas of the ciliated epithelial cells composing the filaments, concentrated the highest mitochondrial densities (MTK Deep Red 633 staining) and the most acidic pH values (as observed with ageladine-a). These mitochondria have low (depolarized) membrane potentials (D psi m) (JC-1 staining), suggesting that the high amounts of ATP required for ciliary beating may be in part produced by non-mitochondrial mechanisms, such as the enzymatic activity of an ATP-regenerating kinase. Nitric oxide (NO, DAF-2DA staining) produced in the region of the peripheral mitochondria may have an effect on mitochondrial electron transport and possibly cause the low membrane potential. High DAF-2DA staining was moreover observed in the muscle cells composing the wall of the blood vessels where NO may be involved in regulating blood vessel diameter. On the ventral bend of the gills, subepithelial mucus glands (SMG) contain large mucous vacuoles showing higher fluorescence intensities for O2.- (DHE staining) than the rest of the tissue. Given the antimicrobial properties of superoxide, release of O2.- into the mucus may help to avoid the development of microbial biofilms on the gill surface. However, cells of the ventral bends are paying a price for this antimicrobial protection, since they show significantly higher oxidative damage, according to the antioxidant enzyme activities and the carbonyl levels, than the rest of the gill tissue. This study provides the first evidence that one single epithelial cell may contain mitochondria with significantly different membrane potentials. Furthermore, we provide new insight into ROS and RNS formation in ex-vivo gill tissues which opens new perspectives for unraveling the different ecophysiological roles of ROS and RNS in multifunctional organs such as gills.

Composition of rocks and minerals from the Bolshoy Lyakhovsky Island (New Siberian Islands)

In the southeast of the Bolshoi Lyakhovsky Island there are outcrops of tectonic outliers composed of low-K medium-Ti tholeiitic basic rocks represented by low altered pillow basalts, as well as by their metamorphosed analogs: amphibolites and blueschists. The rocks are depleted in light rare-earth elements and were melted out of a depleted mantle source enriched in Th, Nb, and Zr also contributed to the rock formation. The magma sources were not affected by subduction-related fluids or melts. The rocks were part of the Jurassic South Anyui ocean basin crust. The blueschists are the crust of the same basin submerged beneath the more southern Anyui-Svyatoi Nos arc to depth of 30-40 km. Pressure and temperature of metamorphism suggest a setting of "warm" subduction. Mineral assemblages of the blueschists record time of a collision of the Anyui-Svyatoi Nos island arc and the New Siberian continental block expressed as a counter-clockwise PT trend. The pressure jump during the collision corresponds to heaping of tectonic covers above the zone of convergence 12 km in total thickness. Ocean rocks were thrust upon the margin of the New Siberian continental block in late Late Jurassic - early Early Cretaceous and mark the NW continuation of the South Anyui suture, one of the main tectonic sutures of the Northeastern Asia.

Stable carbon and oxygen isotope record of diagenetic carbonates from the New Jersey shelf

Carbon cycling is an important but poorly understood process on passive continental margins. In this study, we use the ionic and stable isotopic composition of interstitial waters and the petrology, mineralogy, and stable isotopic composition of authigenic carbonates collected from Ocean Drilling Program (ODP) Leg 174A (Sites 1071 and 1072) to constrain the origin of the carbonates and the evolution of methane on the outer New Jersey shelf. The pore fluids of the New Jersey continental shelf are characterized by (1) a fresh-brackish water plume, and (2) organic matter degradation reactions, which proceed through sulfate reduction. However, only minor methanogenesis occurs. The oxygen isotopic composition of the pore fluids supports a meteoric origin of the low salinity fluids. Authigenic carbonates are found in nodules, thin (~1-cm) layers, and carbonate cemented pavements. Siderite is the most common authigenic carbonate, followed by dolomite and calcite. The oxygen isotopic composition of the authigenic carbonates, i.e. 1.3-6.5 per mil PeeDee Belemnite (PDB), indicates an origin in marine pore fluids. The carbon isotopic composition of dolomite cements range from -16.4 to -8.8 per mil PDB, consistent with formation within the zone of sulfate reduction. Siderite d13C values show a greater range (-17.67-16.4 per mil), but are largely positive (mean=2.8 per mil) and are interpreted to have formed throughout the zone of methanogenesis. In contrast, calcite d13C values are highly negative (as low as -41.7 per mil)and must have formed from waters with a large component of dissolved inorganic carbon derived from methane oxidation. Pore water data show that despite complete sulfate reduction, methanogenesis appears not to be an important process presently occurring in the upper 400 m of the outer New Jersey shelf. In contrast, the carbon isotopic composition of the siderites and calcites document an active methanogenic zone during their formation. The methane may have been either oxidized or vented from shelf sediments, perhaps during sea-level fluctuations. If this unaccounted and variable methane flux is an areally important process during Neogene sea-level fluctuations, then it likely plays an important role in long-term carbon cycling on passive continental margins

Neogene and Paleocene-Maestrichtian calcareous nannofossils in sediments of the upper continental rise off New Jersey

Maestrichtian to Holocene calcareous nannofossils from two closely spaced sites on the upper continental rise some 100 miles (161 km) southeast of Atlantic City, New Jersey, were zoned in order to help date a major canyon-cutting event in the late Miocene and to delineate and correlate other hiatuses with seismic stratigraphy. Mid-middle Eocene through middle Miocene sediments (Zones CP14 to CN6) were not recovered in these holes, but nearly all other zones are accounted for. The Eocene section is described in a companion chapter (Applegate and Wise, 1987, doi:10.2973/dsdp.proc.93.118.1987). Nannofossils are generally sparse and moderately preserved in the clastic sediments of Site 604. Sedimentation rates are extremely high for the upper Pleistocene (201 m/m.y. minimum) above a hiatus calculated to span 0.44 to 1.1 Ma. The associated disconformity is correlated with local seismic reflection Horizon Pr . Sedimentation rates continue to be high (93 m/m.y.) down to a second hiatus in the upper Pliocene dated from about 2.4 to 2.9 (or possibly 3.3) Ma. The disconformity associated with this hiatus is correlated with local seismic reflection Horizon P2 and regional Reflector Blue, which can be interpreted to mark either the onset of Northern Hemisphere continental glaciation or circulation changes associated with the closure of the Central American Seaway. Sedimentation rates in the pre-glacial lower Pliocene are only about a third those in the glacial upper Pliocene. A prominent disconformity in the upper Miocene marks a major lithologic boundary that separates Messinian(?) glauconitic claystones above from lower Tortonian conglomeratic debris flows and turbidites below. The debris flows recovered are assigned to nannofossil Zones CN8a and CN7, but drilling difficulties prevented penetration of the bottom of this sequence some 100 m below the terminal depth of the hole. Correlation of the lower bounding seismic reflector (M2/Merlin?) to a drift sequence drilled on the lower rise at DSDP Site 603, however, predicts that the debris flows began close to the beginning of the late Miocene (upper Zone CN6 time) at about 10.5 Ma. The debris flows represent a major canyon-cutting event that we correlate with the beginning of the particularly severe late Miocene glaciations believed to be associated with the formation of the West Antarctic Ice Sheet. The existence of these spectacular debris flows strongly suggest that the late Miocene glacio-eustatic low stand occurred during Vail Cycle TM3.1 (lower Tortonian) rather than during Vail Cycle TM3.2 (Messinian) as originally published. Beneath a set of coalesced regional disconformities centered upon seismic reflection Horizon Au, coccoliths are abundant and in general are moderately preserved at Site 605 in a 619-m carbonate section extending from the middle Eocene Zone CP13b to the upper Maestrichtian Lithraphidites quadratus Zone. Sedimentation rates are 37 m/m.y. in the Eocene down to a condensed interval near the base (Zone CP9). A disconformity is suspected near the Eocene/Paleocene boundary. Sedimentation rates for the upper Paleocene Zone CP8 are similar to those of the Eocene, but Zones CP7 and CP6 lie within another condensed interval. The highest Paleocene rates are 67 m/m.y. down through Zones CP5 and CP4 to a major disconformity that separates the upper Paleocene from the Danian. This hiatus spans about 2.6 m.y. (upper Zone CP3 to lower Zone CP2) and corresponds to the major sea-level drop at the base of Vail Cycle TE2.1. As the most prominent break in this Paleogene section, it may correspond to seismic reflection Horizon A* of the North American Basin. Sedimentation rates from this point to the Cretaceous/Tertiary boundary drop to 11 m/m.y., still high for a Paleocene DSDP section. No major break in deposition could be detected at the Cretaceous/Tertiary boundary.

Deep water formation in the North Pacific and deglacial CO2 rise

Deep water formation in the North Atlantic and Southern Ocean is widely thought to influence deglacial CO2 rise and climate change; here we suggest that deep water formation in the North Pacific may also play an important role. We present paired radiocarbon and boron isotope data from foraminifera from sediment core MD02-2489 at 3640 m in the North East Pacific. These show a pronounced excursion during Heinrich Stadial 1, with benthic-planktic radiocarbon offsets dropping to ~350 years, accompanied by a decrease in benthic d11B. We suggest this is driven by the onset of deep convection in the North Pacific, which mixes young shallow waters to depth, old deep waters to the surface, and low-pH water from intermediate depths into the deep ocean. This deep water formation event was likely driven by an increase in surface salinity, due to subdued atmospheric/monsoonal freshwater flux during Heinrich Stadial 1. The ability of North Pacific Deep Water (NPDW) formation to explain the excursions seen in our data is demonstrated in a series of experiments with an intermediate complexity Earth system model. These experiments also show that breakdown of stratification in the North Pacific leads to a rapid ~30 ppm increase in atmospheric CO2, along with decreases in atmospheric d13C and D14C, consistent with observations of the early deglaciation. Our inference of deep water formation is based mainly on results from a single sediment core, and our boron isotope data are unavoidably sparse in the key HS1 interval, so this hypothesis merits further testing. However we note that there is independent support for breakdown of stratification in shallower waters during this period, including a minimum in d15N, younging in intermediate water 14C, and regional warming. We also re-evaluate deglacial changes in North Pacific productivity and carbonate preservation in light of our new data, and suggest that the regional pulse of export production observed during the Bølling-Allerød is promoted by relatively stratified conditions, with increased light availability and a shallow, potent nutricline. Overall, our work highlights the potential of NPDW formation to play a significant and hitherto unrealized role in deglacial climate change and CO2 rise.

Stable carbon isotope record of benthic foraminifera across the Paleocene-Eocene thermal maximum in the New Jersey Coastal Plain

In the New Jersey Coastal Plain, a silty to clayey sedimentary unit (the Marlboro Formation) represents deposition during the Paleocene-Eocene thermal maximum (PETM). This interval is remarkably different from the glauconitic sands and silts of the underlying Paleocene Vincentown and overlying Eocene Manasquan Formation. We integrate new and published stable isotope, biostratigraphic, lithostratigraphic and ecostratigraphic records, constructing a detailed time frame for the PETM along a depth gradient at core sites Clayton, Wilson Lake, Ancora and Bass River (updip to downdip). The onset of the PETM, marked by the base of the carbon isotope excursion (CIE), is within the gradual transition from glauconitic silty sands to silty clay, and represented fully at the updip sites (Wilson Lake and Clayton). The CIE "core" interval is expanded at the updip sites, but truncated. The CIE "core" is complete at the Bass River and Ancora sites, where the early part of the recovery is present (most complete at Ancora). The extent to which the PETM is expressed in the sediments is highly variable between sites, with a significant unconformity at the base of the overlying lower Eocene sediments. Our regional correlation framework provides an improved age model, allowing better understanding of the progression of environmental changes during the PETM. High-resolution benthic foraminiferal data document the change from a sediment-starved shelf setting to a tropical, river-dominated mud-belt system during the PETM, probably due to intensification of the hydrologic cycle. The excellent preservation of foraminifera during the PETM and the lack of severe benthic extinction suggest there was no extreme ocean acidification in shelf settings.