Reduced IFNλ4 activity is associated with improved HCV clearance and reduced expression of interferon-stimulated genes

Hepatitis C virus (HCV) infections are the major cause of chronic liver disease, cirrhosis and hepatocellular carcinoma worldwide. Both spontaneous and treatment-induced clearance of HCV depend on genetic variation within the interferon-lambda locus, but until now no clear causal relationship has been established. Here we demonstrate that an amino-acid substitution in the IFNλ4 protein changing a proline at position 70 to a serine (P70S) substantially alters its antiviral activity. Patients harbouring the impaired IFNλ4-S70 variant display lower interferon-stimulated gene (ISG) expression levels, better treatment response rates and better spontaneous clearance rates, compared with patients coding for the fully active IFNλ4-P70 variant. Altogether, these data provide evidence supporting a role for the active IFNλ4 protein as the driver of high hepatic ISG expression as well as the cause of poor HCV clearance.

Competitive fitness in coronaviruses is not correlated with size or number of double-membrane vesicles under reduced-temperature growth conditions.

Positive-stranded viruses synthesize their RNA in membrane-bound organelles, but it is not clear how this benefits the virus or the host. For coronaviruses, these organelles take the form of double-membrane vesicles (DMVs) interconnected by a convoluted membrane network. We used electron microscopy to identify murine coronaviruses with mutations in nsp3 and nsp14 that replicated normally while producing only half the normal amount of DMVs under low-temperature growth conditions. Viruses with mutations in nsp5 and nsp16 produced small DMVs but also replicated normally. Quantitative reverse transcriptase PCR (RT-PCR) confirmed that the most strongly affected of these, the nsp3 mutant, produced more viral RNA than wild-type virus. Competitive growth assays were carried out in both continuous and primary cells to better understand the contribution of DMVs to viral fitness. Surprisingly, several viruses that produced fewer or smaller DMVs showed a higher fitness than wild-type virus at the reduced temperature, suggesting that larger and more numerous DMVs do not necessarily confer a competitive advantage in primary or continuous cell culture. For the first time, this directly demonstrates that replication and organelle formation may be, at least in part, studied separately during infection with positive-stranded RNA virus. IMPORTANCE The viruses that cause severe acute respiratory syndrome (SARS), poliomyelitis, and hepatitis C all replicate in double-membrane vesicles (DMVs). The big question about DMVs is why they exist in the first place. In this study, we looked at thousands of infected cells and identified two coronavirus mutants that made half as many organelles as normal and two others that made typical numbers but smaller organelles. Despite differences in DMV size and number, all four mutants replicated as efficiently as wild-type virus. To better understand the relative importance of replicative organelles, we carried out competitive fitness experiments. None of these viruses was found to be significantly less fit than wild-type, and two were actually fitter in tests in two kinds of cells. This suggests that viruses have evolved to have tremendous plasticity in the ability to form membrane-associated replication complexes and that large and numerous DMVs are not exclusively associated with efficient coronavirus replication.

Synthesis of cyclopentane amide DNA (cpa-DNA) and its pairing properties

We recently reported on the synthesis and pairing properties of the DNA analogue bicyclo[3.2.1]amide DNA (bca-DNA). In this analogue the nucleobases are attached via a linear, 4-bond amide-linker to a structurally preorganized sugar-phosphate backbone unit. To define the importance of the degree of structural rigidity of the bca-backbone unit on the pairing properties, we designed the structurally simpler cyclopentane amide DNA (cpa-DNA), in which the bicyclo[3.2.1]-scaffold was reduced to a cyclopentane unit while the base-linker was left unchanged. Here we present a synthetic route to the enantiomerically pure cpa-DNA monomers and the corresponding phosphoramidites containing the bases A and T, starting from a known, achiral precursor in 9 and 12 steps, respectively. Fully modified oligodeoxynucleotides were synthesized by standard solid-phase oligonucleotide chemistry, and their base-pairing properties with complementary oligonucleotides of the DNA-, RNA-, bca-DNA-, and cpa-DNA-backbones were assessed by UV melting curves and CD-spectroscopic methods. We found that cpa-oligoadenylates form duplexes with complementary DNA that are less stable by -2.7 degrees C/mod. compared to DNA. The corresponding cpa-oligothymidylates do not participate in complementary base-pairing with any of the investigated backbone systems except with its own (homo-duplex). As its congener bca-DNA, cpa-DNA seems to prefer left-handed helical duplex structures with DNA or with itself as indicated by the CD spectra

A naturally occurring prfA truncation in a Listeria monocytogenes field strain contributes to reduced replication and cell-to-cell spread

Listeria (L.) monocytogenes is an environmental bacterium that may become an intracellular pathogen upon ingestion to cause gastroenteritis, septicaemia, abortions, and/or fatal infections of the central nervous system. We here describe a L. monocytogenes field strain (JF5171) isolated from a bovine placenta in the context of abortion, which exhibited attenuation in bovine brain-slice cultures. The whole genome of strain JF5171 was sequenced, and the invasion, replication, and intercellular spread of JF5171 were further analyzed by quantification of colony forming units and immunofluorescence studies. Phospholipase and hemolysis activity of JF5171 were also quantified along with transcription levels of actA, hly and prfA. The data obtained were compared to those of the widely used L. monocytogenes reference strain, EGD-e. JF5171 exhibited reduced replication and lower levels of phospholipase and hemolysis activity. Invasion and cell-to-cell spread was strongly decreased compared to EGD-e, and actin polymerization was absent. A frame shift deletion was identified in the JF5171 coding region of the major regulator for virulence, prfA. This resulted in a truncated C-terminus sequence (WEN* vs. WGKLN*). In addition, a point mutation resulted in a lysine to arginine substitution at amino acid position 197. Complementation with prfA from EGD-e and with (EGD-e) prfA-K197N increased the replication and spread efficiency of JF5171. In contrast, complementation with the truncated version of prfA had no effect. Taken together, these results suggest that the truncated C-terminus of prfA considerably contributes to the strongly attenuated phenotype observed in vitro.

New perioperative fluid and pharmacologic management protocol results in reduced blood loss, faster return of bowel function, and overall recovery.

Cystectomy and urinary diversion have high morbidity, and strategies to reduce complications are of utmost importance. Epidural analgesia and optimized fluid management are considered key factors contributing to successful enhanced recovery after surgery. In colorectal surgery, there is strong evidence that an intraoperative fluid management aiming for a postoperative zero fluid balance results in lower morbidity including a faster return of bowel function. Recently, a randomized clinical trial focusing on radical cystectomy demonstrated that a restrictive intraoperative hydration combined with a concomitant administration of norepinephrine reduced intraoperative blood loss, the need for blood transfusion and morbidity. The purpose of this review is to highlight specific anesthesiological aspects which have been shown to improve outcome after RC with urinary diversion.

Zinc supplementation reduced DNA breaks in Ethiopian women.

Assessment of zinc status remains a challenge largely because serum/plasma zinc may not accurately reflect an individual's zinc status. The comet assay, a sensitive method capable of detecting intracellular DNA strand breaks, may serve as a functional biomarker of zinc status. We hypothesized that effects of zinc supplementation on intracellular DNA damage could be assessed from samples collected in field studies in Ethiopia using the comet assay. Forty women, from villages where reported consumption of meat was less than once per month and phytate levels were high, received 20 mg zinc as zinc sulfate or placebo daily for 17 days in a randomized placebo-controlled trial. Plasma zinc concentrations were determined by inductively coupled plasma mass spectrometry. Cells from whole blood at the baseline and end point of the study were embedded in agarose, electrophoresed, and stained before being scored by an investigator blinded to the treatments. Although zinc supplementation did not significantly affect plasma zinc, mean (± SEM) comet tail moment measurement of supplemented women decreased from 39.7 ± 2.7 to 30.0 ± 1.8 (P< .005), indicating a decrease in DNA strand breaks in zinc-supplemented individuals. These findings demonstrated that the comet assay could be used as a functional assay to assess the effects of zinc supplementation on DNA integrity in samples collected in a field setting where food sources of bioavailable zinc are limited. Furthermore, the comet assay was sufficiently sensitive to detect changes in zinc status as a result of supplementation despite no significant changes in plasma zinc.

Optimal Confidence Bands for Shape-Restricted Curves

Let Y be a stochastic process on [0,1] satisfying dY(t)=n 1/2 f(t)dt+dW(t) , where n≥1 is a given scale parameter (`sample size'), W is standard Brownian motion and f is an unknown function. Utilizing suitable multiscale tests, we construct confidence bands for f with guaranteed given coverage probability, assuming that f is isotonic or convex. These confidence bands are computationally feasible and shown to be asymptotically sharp optimal in an appropriate sense.

On the reduced sensitivity of the Atlantic overturning to Greenland ice sheet melting in projections: a multi-model assessment

Large uncertainties exist concerning the impact of Greenland ice sheet melting on the Atlantic meridional overturning circulation (AMOC) in the future, partly due to different sensitivity of the AMOC to freshwater input in the North Atlantic among climate models. Here we analyse five projections from different coupled ocean–atmosphere models with an additional 0.1 Sv (1 Sv = 10 6 m3/s) of freshwater released around Greenland between 2050 and 2089. We find on average a further weakening of the AMOC at 26°N of 1.1 ± 0.6 Sv representing a 27 ± 14% supplementary weakening in 2080–2089, as compared to the weakening relative to 2006–2015 due to the effect of the external forcing only. This weakening is lower than what has been found with the same ensemble of models in an identical experimen - tal set-up but under recent historical climate conditions. This lower sensitivity in a warmer world is explained by two main factors. First, a tendency of decoupling is detected between the surface and the deep ocean caused by an increased thermal stratification in the North Atlantic under the effect of global warming. This induces a shoaling of ocean deep ventilation through convection hence ventilating only intermediate levels. The second important effect concerns the so-called Canary Current freshwater leakage; a process by which additionally released fresh water in the North Atlantic leaks along the Canary Current and escapes the convection zones towards the subtropical area. This leakage is increasing in a warming climate, which is a consequence of decreasing gyres asymmetry due to changes in Ekman rumping. We suggest that these modifications are related with the northward shift of the jet stream in a warmer world. For these two reasons the AMOC is less susceptible to freshwater perturbations (near the deep water formation sides) in the North Atlantic as compared to the recent historical climate conditions. Finally, we propose a bilinear model that accounts for the two former processes to give a conceptual explanation about the decreasing AMOC sensitivity due to freshwater input. Within the limit of this bilinear model, we find that 62 ± 8% of the reduction in sensitivity is related with the changes in gyre asymmetry and freshwater leakage and 38 ± 8% is due to the reduction in deep ocean ventilation associated with the increased stratification in the North Atlantic.

Impact of a Reduced Arctic Sea Ice Cover on Ocean and Atmospheric Properties

The Arctic sea ice cover declined over the last few decades and reached a record minimum in 2007, with a slight recovery thereafter. Inspired by this the authors investigate the response of atmospheric and oceanic properties to a 1-yr period of reduced sea ice cover. Two ensembles of equilibrium and transient simulations are produced with the Community Climate System Model. A sea ice change is induced through an albedo change of 1 yr. The sea ice area and thickness recover in both ensembles after 3 and 5 yr, respectively. The sea ice anomaly leads to changes in ocean temperature and salinity to a depth of about 200 m in the Arctic Basin. Further, the salinity and temperature changes in the surface layer trigger a “Great Salinity Anomaly” in the North Atlantic that takes roughly 8 yr to travel across the North Atlantic back to high latitudes. In the atmosphere the changes induced by the sea ice anomaly do not last as long as in the ocean. The response in the transient and equilibrium simulations, while similar overall, differs in specific regional and temporal details. The surface air temperature increases over the Arctic Basin and the anomaly extends through the whole atmospheric column, changing the geopotential height fields and thus the storm tracks. The patterns of warming and thus the position of the geopotential height changes vary in the two ensembles. While the equilibrium simulation shifts the storm tracks to the south over the eastern North Atlantic and Europe, the transient simulation shifts the storm tracks south over the western North Atlantic and North America. The authors propose that the overall reduction in sea ice cover is important for producing ocean anomalies; however, for atmospheric anomalies the regional location of the sea ice anomalies is more important. While observed trends in Arctic sea ice are large and exceed those simulated by comprehensive climate models, there is little evidence based on this particular model that the seasonal loss of sea ice (e.g., as occurred in 2007) would constitute a threshold after which the Arctic would exhibit nonlinear, irreversible, or strongly accelerated sea ice loss. Caution should be exerted when extrapolating short-term trends to future sea ice behavior.

Topology of the ambient boundary and the convergence of causal curves

We discuss the topological nature of the boundary spacetime, the conformal infinity of the ambient cosmological metric. Due to the existence of a homothetic group, the bounding spacetime must be equipped not with the usual Euclidean metric topology but with the Zeeman fine topology. This then places severe constraints to the convergence of a sequence of causal curves and the extraction of a limit curve, and also to our ability to formulate conditions for singularity formation.

Estimation of reference curves for the urinary steroid metabolome in the first year of life in healthy children: tracing the complexity of human postnatal steroidogenesis

CONTEXT Complex steroid disorders such as P450 oxidoreductase deficiency or apparent cortisone reductase deficiency may be recognized by steroid profiling using chromatographic mass spectrometric methods. These methods are highly specific and sensitive, and provide a complete spectrum of steroid metabolites in a single measurement of one sample which makes them superior to immunoassays. The steroid metabolome during the fetal-neonatal transition is characterized by a) the metabolites of the fetal-placental unit at birth, b) the fetal adrenal androgens until its involution 3-6 months postnatally, and c) the steroid metabolites produced by the developing endocrine organs. All these developmental events change the steroid metabolome in an age- and sex-dependent manner during the first year of life. OBJECTIVE The aim of this study was to provide normative values for the urinary steroid metabolome of healthy newborns at short time intervals in the first year of life. METHODS We conducted a prospective, longitudinal study to measure 67 urinary steroid metabolites in 21 male and 22 female term healthy newborn infants at 13 time-points from week 1 to week 49 of life. Urine samples were collected from newborn infants before discharge from hospital and from healthy infants at home. Steroid metabolites were measured by gas chromatography-mass spectrometry (GC-MS) and steroid concentrations corrected for urinary creatinine excretion were calculated. RESULTS 61 steroids showed age and 15 steroids sex specificity. Highest urinary steroid concentrations were found in both sexes for progesterone derivatives, in particular 20α-DH-5α-DH-progesterone, and for highly polar 6α-hydroxylated glucocorticoids. The steroids peaked at week 3 and decreased by ∼80% at week 25 in both sexes. The decline of progestins, androgens and estrogens was more pronounced than of glucocorticoids whereas the excretion of corticosterone and its metabolites and of mineralocorticoids remained constant during the first year of life. CONCLUSION The urinary steroid profile changes dramatically during the first year of life and correlates with the physiologic developmental changes during the fetal-neonatal transition. Thus detailed normative data during this time period permit the use of steroid profiling as a powerful diagnostic tool.