Dengue virus NS5 is the most highly conserved amongst the viral non-structural proteins and is responsible for capping, methylation and replication of the flavivirus RNA genome. Interactions of NS5 ...with host proteins also modulate host immune responses. Although replication occurs in the cytoplasm, an unusual characteristic of DENV2 NS5 is that it localizes to the nucleus during infection with no clear role in replication or pathogenesis. We examined NS5 of DENV1 and 2, which exhibit the most prominent difference in nuclear localization, employing a combination of functional and structural analyses. Extensive gene swapping between DENV1 and 2 NS5 identified that the C-terminal 18 residues (Cter18) alone was sufficient to direct the protein to the cytoplasm or nucleus, respectively. The low micromolar binding affinity between NS5 Cter18 and the nuclear import receptor importin-alpha (Impα), allowed their molecular complex to be purified, crystallised and visualized at 2.2 Å resolution using x-ray crystallography. Structure-guided mutational analysis of this region in GFP-NS5 clones of DENV1 or 2 and in a DENV2 infectious clone reveal residues important for NS5 subcellular localization. Notably, the trans conformation adopted by Pro-884 allows proper presentation for binding Impα and mutating this proline to Thr, as present in DENV1 NS5, results in mislocalizaion of NS5 to the cytoplasm without compromising virus fitness. In contrast, a single mutation to alanine at NS5 position R888, a residue conserved in all flaviviruses, resulted in a completely non-viable virus, and the R888K mutation led to a severely attenuated phentoype, even though NS5 was located in the nucleus. R888 forms a hydrogen bond with Y838 that is also conserved in all flaviviruses. Our data suggests an evolutionarily conserved function for NS5 Cter18, possibly in RNA interactions that are critical for replication, that is independent of its role in subcellular localization.
High purity bright Sn, Sn–Cu and Sn–Pb layers, 3, 7 and 16
μm thick were electrodeposited on phosphor bronze cantilever beams in a rotating disk apparatus. Beam deflection measurements within 15
min ...of plating proved that all electrodeposits had in-plane compressive stress. In several days, the surfaces of the Sn–Cu deposits, which have the highest compressive stress, develop 50
μm contorted hillocks and 200
μm whiskers, pure Sn deposits develop 20
μm compact conical hillocks, and Sn–Pb deposits, which have the lowest compressive stress, remain unchanged. The differences between the initial compressive stresses for each alloy and pure Sn is due to the rapid precipitation of Cu
6Sn
5 or Pb particles, respectively, within supersaturated Sn grains produced by electrodeposition. Over longer time, analysis of beam deflection measurements indicates that the compressive stress is augmented by the formation of Cu
6Sn
5 on the bronze/Sn interface, while creep of the electrodeposit tends to decrease the compressive stress. Uniform creep occurs for Sn–Pb because it has an equi-axed grain structure. Localized creep in the form of hillocks and whiskers occurs for Sn and Sn–Cu because both have columnar structures. Compact hillocks form for the Sn deposits because the columnar grain boundaries are mobile. Contorted hillocks and whiskers form for the Sn–Cu deposits because the columnar grain boundary motion is impeded.
Abstract Purpose Studies regarding the effects of aesthetic outcomes after breast cancer surgery on quality of life (QoL) have yielded inconsistent results. This study analyzed the aesthetic outcomes ...and QoL of women who underwent breast conserving surgery (BCS) or total mastectomy with immediate reconstruction (TMIR) using objective and validated methods. Patients and methods QoL questionnaires (EORTC QLQ-C30, BR23, and HADs) were administered at least 1 year after surgery and adjuvant therapy to 485 patients who underwent BCS, 46 who underwent TMIR, and 87 who underwent total mastectomy (TM) without reconstruction. Aesthetic results were evaluated using BCCT.core software and by a panel of physicians. Patients' body image perception was assessed using the body image scale (BIS). Results QoL outcomes, including for social and role functioning, fatigue, pain, body image, and arm symptoms, were significantly better in the BCS and TMIR groups than in the TM group (p<0.05 each). BIS was significantly better in the BCS than in the TM or TMIR group (p<0.001 each). In the BCS and TMIR groups, general QoL factors were not significantly associated with objective cosmetic outcomes, except for body image in the QLQ-BR23. In contrast, patients with poorer BIS score reported lower QoL in almost all items of the QLQ-C30, BR23, and HADS (p<0.05 each). Conclusion In conclusion, BCS and TMIR enhanced QoL compared with TM. Among BCS and TMIR patients, objectively measured cosmetic results did not affect general QoL. S elf-perception of body image seems to be more important for QoL after breast cancer surgery.
Sn-rich alloys in the Sn-Ag-Cu system are being studied for their potential as Pb-free solders. Thus, the location of the ternary eutectic involving L, (Sn), Ag sub(3)Sn and Cu sub(6)Sn sub(5) phases ...is of critical interest. Phase diagram data in the Sn-rich corner of the Sn-Ag-Cu system are measured. The ternary eutectic is confirmed to be at a composition of 3.5 wt.% Ag, 0.9 wt.% Cu at a temperature of 217.2 plus or minus 0.2 degree C (2 sigma ). A thermodynamic calculation of the Sn-rich part of the diagram from the three constituent binary systems and the available ternary data using the CALPHAD method is conducted. The best fit to the experimental data is 3.66 wt.% Ag and 0.91 wt.% Cu at a temperature of 216.3 degree C. Using the thermodynamic description to obtain the enthalpy-temperature relation, the DTA signal is simulated and used to explain the difficulty of liquidus measurements in these alloys.
While interstitial fibrosis plays a significant role in heart failure, our understanding of disease progression in humans is limited. To address this limitation, we have engineered a ...cardiac-fibrosis-on-a-chip model consisting of a microfabricated device with live force measurement capabilities using co-cultured human cardiac fibroblasts and pluripotent stem cell-derived cardiomyocytes. Transforming growth factor-β was used as a trigger for fibrosis. Here, we have reproduced the classic hallmarks of fibrosis-induced heart failure including high collagen deposition, increased tissue stiffness, BNP secretion, and passive tension. Force of contraction was significantly decreased in fibrotic tissues that displayed a transcriptomic signature consistent with human cardiac fibrosis/heart failure. Treatment with an anti-fibrotic drug decreased tissue stiffness and BNP secretion, with corresponding changes in the transcriptomic signature. This model represents an accessible approach to study human heart failure in vitro, and allows for testing anti-fibrotic drugs while facilitating the real-time assessment of cardiomyocyte function.
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Summary Objectives Symptomatic degenerative disc disease (DDD) is associated with neovascularization and nerve ingrowth into intervertebral discs (IVDs). Notochordal cells (NCs) are key cells that ...may lead to regeneration of IVDs. However, their activities under conditions of hypoxia, the real environment of IVD, are not well known. We hypothesized that NCs may inhibit neovascularization by interacting with endothelial cells (ECs) under hypoxia. Design Human IVDs were isolated and cultured to produce nucleus pulposus (NP) cell conditioned medium (NPCM). Immortalized human microvascular ECs were cultured in NPCM with notochordal cell-rich rabbit nucleus pulposus cells (rNC) under hypoxia. Vascular endothelial growth factor (VEGF), vascular cell adhesion molecule (VCAM), and interleukin-8 (IL-8) were analyzed by ELISA. Focal adhesion kinase (FAK), filamentous actin (F-actin), and platelet-derived growth factor (PDGF) were evaluated to investigate EC activity. Wound-healing migration assays were performed to examine EC migration. Results The VEGF level of EC cells cultured in NPCM was significantly higher under hypoxia compared to normoxia. VEGF expression was significantly decreased, and FAK, F-actin, PDGF expression were inhibited when ECs were cocultured with rNCs under hypoxia. ECs cocultured with rNC in NPCM showed significantly decreased migratory activity compared to those without rNC under hypoxia. Conclusions The angiogenic capacity of ECs was significantly inhibited by NCs under hypoxia via a VEGF-related pathway. Our results suggest that NCs may play a key role in the development of IVDs by inhibiting vascular growth within the disc, and this may be a promising novel therapeutic strategy for targeting vascular ingrowth in symptomatic DDD.
Intracerebral haemorrhage growth is associated with poor clinical outcome and is a therapeutic target for improving outcome. We aimed to determine the absolute risk and predictors of intracerebral ...haemorrhage growth, develop and validate prediction models, and evaluate the added value of CT angiography.
In a systematic review of OVID MEDLINE—with additional hand-searching of relevant studies' bibliographies— from Jan 1, 1970, to Dec 31, 2015, we identified observational cohorts and randomised trials with repeat scanning protocols that included at least ten patients with acute intracerebral haemorrhage. We sought individual patient-level data from corresponding authors for patients aged 18 years or older with data available from brain imaging initially done 0·5–24 h and repeated fewer than 6 days after symptom onset, who had baseline intracerebral haemorrhage volume of less than 150 mL, and did not undergo acute treatment that might reduce intracerebral haemorrhage volume. We estimated the absolute risk and predictors of the primary outcome of intracerebral haemorrhage growth (defined as >6 mL increase in intracerebral haemorrhage volume on repeat imaging) using multivariable logistic regression models in development and validation cohorts in four subgroups of patients, using a hierarchical approach: patients not taking anticoagulant therapy at intracerebral haemorrhage onset (who constituted the largest subgroup), patients taking anticoagulant therapy at intracerebral haemorrhage onset, patients from cohorts that included at least some patients taking anticoagulant therapy at intracerebral haemorrhage onset, and patients for whom both information about anticoagulant therapy at intracerebral haemorrhage onset and spot sign on acute CT angiography were known.
Of 4191 studies identified, 77 were eligible for inclusion. Overall, 36 (47%) cohorts provided data on 5435 eligible patients. 5076 of these patients were not taking anticoagulant therapy at symptom onset (median age 67 years, IQR 56–76), of whom 1009 (20%) had intracerebral haemorrhage growth. Multivariable models of patients with data on antiplatelet therapy use, data on anticoagulant therapy use, and assessment of CT angiography spot sign at symptom onset showed that time from symptom onset to baseline imaging (odds ratio 0·50, 95% CI 0·36–0·70; p<0·0001), intracerebral haemorrhage volume on baseline imaging (7·18, 4·46–11·60; p<0·0001), antiplatelet use (1·68, 1·06–2·66; p=0·026), and anticoagulant use (3·48, 1·96–6·16; p<0·0001) were independent predictors of intracerebral haemorrhage growth (C-index 0·78, 95% CI 0·75–0·82). Addition of CT angiography spot sign (odds ratio 4·46, 95% CI 2·95–6·75; p<0·0001) to the model increased the C-index by 0·05 (95% CI 0·03–0·07).
In this large patient-level meta-analysis, models using four or five predictors had acceptable to good discrimination. These models could inform the location and frequency of observations on patients in clinical practice, explain treatment effects in prior randomised trials, and guide the design of future trials.
UK Medical Research Council and British Heart Foundation.
Despite theoretical predictions, it remains an experimental challenge to realize an artificial magnetic skyrmion whose topology can be well controlled and tailored so that its topological effect can ...be revealed explicitly in a deformation of the spin textures. Here we report epitaxial magnetic thin films in which an artificial skyrmion is created by embedding a magnetic vortex into an out-of-plane aligned spin environment. By changing the relative orientation between the central vortex core polarity and the surrounding out-of-plane spins, we are able to control and tailor the system between two skyrmion topological states. An in-plane magnetic field is used to annihilate the skyrmion core by converting the central vortex state into a single domain state. Our result shows distinct annihilation behaviour of the skyrmion core for the two different skyrmion states, suggesting a topological effect of the magnetic skyrmions in the core annihilation process.
Dengue virus multifunctional proteins NS3 protease/helicase and NS5 methyltransferase/RNA-dependent RNA polymerase form part of the viral replication complex and are involved in viral RNA genome ...synthesis, methylation of the 5′-cap of viral genome, and polyprotein processing among other activities. Previous studies have shown that NS5 residue Lys-330 is required for interaction between NS3 and NS5. Here, we show by competitive NS3-NS5 interaction ELISA that the NS3 peptide spanning residues 566–585 disrupts NS3-NS5 interaction but not the null-peptide bearing the N570A mutation. Small angle x-ray scattering study on NS3(172–618) helicase and covalently linked NS3(172–618)-NS5(320–341) reveals a rigid and compact formation of the latter, indicating that peptide NS5(320–341) engages in specific and discrete interaction with NS3. Significantly, NS3:Asn-570 to alanine mutation introduced into an infectious DENV2 cDNA clone did not yield detectable virus by plaque assay even though intracellular double-stranded RNA was detected by immunofluorescence. Detection of increased negative-strand RNA synthesis by real time RT-PCR for the NS3:N570A mutant suggests that NS3-NS5 interaction plays an important role in the balanced synthesis of positive- and negative-strand RNA for robust viral replication. Dengue virus infection has become a global concern, and the lack of safe vaccines or antiviral treatments urgently needs to be addressed. NS3 and NS5 are highly conserved among the four serotypes, and the protein sequence around the pinpointed amino acids from the NS3 and NS5 regions are also conserved. The identification of the functionally essential interaction between the two proteins by biochemical and reverse genetics methods paves the way for rational drug design efforts to inhibit viral RNA synthesis.NS3-NS5 interaction is important for the dengue virus life cycle.
NS3 residue Asn-570 is essential for its interaction with NS5; mutation in an infectious cDNA abolished virus production and reduced positive-strand RNA synthesis.
NS3-NS5 interaction may be required for coordinated positive- and negative-strand RNA synthesis.
NS3-NS5 interaction may be a target for rational design of antiviral drugs.
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