Probing the different chaperone activities of the bacterial HSP70-HSP40 system using a thermolabile luciferase substrate.

During mild heat-stress, a native thermolabile polypeptide may partially unfold and transiently expose water-avoiding hydrophobic segments that readily tend to associate into a stable misfolded species, rich in intra-molecular non-native beta-sheet structures. When the concentration of the heat-unfolded intermediates is elevated, the exposed hydrophobic segments tend to associate with other molecules into large stable insoluble complexes, also called "aggregates." In mammalian cells, stress- and mutation-induced protein misfolding and aggregation may cause degenerative diseases and aging. Young cells, however, effectively counteract toxic protein misfolding with a potent network of molecular chaperones that bind hydrophobic surfaces and actively unfold otherwise stable misfolded and aggregated polypeptides. Here, we followed the behavior of a purified, initially mostly native thermolabile luciferase mutant, in the presence or absence of the Escherichia coli DnaK-DnaJ-GrpE chaperones and/or of ATP, at 22 °C or under mild heat-stress. We concomitantly measured luciferase enzymatic activity, Thioflavin-T fluorescence, and light-scattering to assess the effects of temperature and chaperones on the formation, respectively, of native, unfolded, misfolded, and/or of aggregated species. During mild heat-denaturation, DnaK-DnaJ-GrpE+ATP best maintained, although transiently, high luciferase activity and best prevented heat-induced misfolding and aggregation. In contrast, the ATP-less DnaK and DnaJ did not maintain optimal luciferase activity and were less effective at preventing luciferase misfolding and aggregation. We present a model accounting for the experimental data, where native, unfolded, misfolded, and aggregated species spontaneously inter-convert, and in which DnaK-DnaJ-GrpE+ATP specifically convert stable misfolded species into unstable unfolded intermediates.

Intramolecular basis set superposition error effects on the planarity of benzene and other aromatic molecules: A solution to the problem

Recently, the surprising result that ab initio calculations on benzene and other planar arenes at correlated MP2, MP3, configuration interaction with singles and doubles (CISD), and coupled cluster with singles and doubles levels of theory using standard Pople’s basis sets yield nonplanar minima has been reported. The planar optimized structures turn out to be transition states presenting one or more large imaginary frequencies, whereas single-determinant-based methods lead to the expected planar minima and no imaginary frequencies. It has been suggested that such anomalous behavior can be originated by two-electron basis set incompleteness error. In this work, we show that the reported pitfalls can be interpreted in terms of intramolecular basis set superposition error (BSSE) effects, mostly between the C–H moieties constituting the arenes. We have carried out counterpoise-corrected optimizations and frequency calculations at the Hartree–Fock, B3LYP, MP2, and CISD levels of theory with several basis sets for a number of arenes. In all cases, correcting for intramolecular BSSE fixes the anomalous behavior of the correlated methods, whereas no significant differences are observed in the single-determinant case. Consequently, all systems studied are planar at all levels of theory. The effect of different intramolecular fragment definitions and the particular case of charged species, namely, cyclopentadienyl and indenyl anions, respectively, are also discussed

The struggle against creative accounting: Is "true and fair view" part of the problem or part of the solution?

Creative accounting is a growing issue of interest in Spain. In this article we argue that the concept true and fair view can limit or promote the use of creative accounting depending upon its interpretation. We review the range of meanings that true and fair view can take at an international level and compare the experience of the United Kingdom with the Australian one by analysing the use of true and fair view to limit creative accounting. Finally, we suggest lines of action to be considered by the Spanish accounting standards-setting institutions.

Attributes of irrigated rice and soil solution as affected by salinity levels of the water layer

In the State of Rio Grande do Sul, Brazil, flooded rice fields using Patos Lagoon as the source of water for irrigation are subject to be damaged by salinity, since this source is bound to the sea on its southern end. The sensitivity of rice is variable during plant development, being higher in the seedling and reproductive periods. However, there is not enough information about the behavior of plants under salt stress during the course of its development, especially in the vegetative stage. This study evaluated the effect of different levels of salinity of irrigation water on the salinity of soil solution over time and on some plant attributes, during the vegetative stage of rice. The study was conducted in a greenhouse, where seeds of the variety IRGA 424 were sown in pots and irrigated with water with electrical conductivity (ECi) levels of: 0.3, 0.75, 1.5, 3.0 and 4.5 dS m-1; from the tillering initiation (V4) until the panicle initiation (PI). The evaluations made were the electrical conductiviy of soil solution (ECe), the dry biomass of plants and stems, tillering, height and the transpiration of plants. The ECe increased with the ECi over time, and was determined by water transpiration flux in pots. The ECe values at the end of the experiment were high and, in most cases, higher than the critical values for flooded rice. The growth attributes of rice were negatively affected from ECi of 2.0 dS m-1 and ECe of 4.0 dS m-1.

Identification of GPx8 as a novel cellular substrate of the hepatitis C virus NS3-4A protease

Background: The hepatitis C virus (HCV) NS3-4A protease is not only an essential component of the viral replication complex and a prime target for antiviral intervention but also a key player in the persistence and pathogenesis of HCV. It cleaves and thereby inactivates two crucial adaptor proteins in viral RNA sensing and innate immunity (MAVS and TRIF) as well as a phosphatase involved in growth factor signaling (TC-PTP). The aim of this study was to identify novel cellular substrates of the NS3-4A protease and to investigate their role in the life cycle and pathogenesis of HCV. Methods: Cell lines inducibly expressing the NS3-4A protease were analyzed in basal as well as interferon- α -stimulated states by stable isotopic labeling using amino acids in cell culture (SILAC) coupled with protein separation and mass spectrometry. Candidates fulfilling strin- gent criteria for potential substrates or products of the NS3-4A protease were further investigated in different experimental sys- tems as well as in liver biopsies from patients with chronic hep- atitis C. Results: SILAC coupled with protein separation and mass spectrometry yielded > 5000 proteins of which 21 can- didates were selected for further analyses. These allowed us to identify GPx8, a membrane-associated peroxidase involved in disulfide bond formation in the endoplasmic reticulum, as a novel cellular substrate of the HCV NS3-4A protease. Cleavage occurs at cysteine in position 11, removing the cytosolic tip of GPx8, and was observed in different experimental systems as well as in liver biopsies from patients with chronic hepatitis C. Further functional studies, involving overexpression and RNA silencing, revealed that GPx8 is a proviral factor involved in viral particle production but not in HCV entry or RNA replica- tion. Conclusions: GPx8 is a proviral host factor cleaved by the HCV NS3-4A protease. Studies investigating the consequences of cleavage for GPx8 function are underway. The identification of novel cellular substrates of the HCV NS3-4A protease should yield new insights into the HCV life cycle and the pathogenesis of hepatitis C and may reveal novel angles for therapeutic inter- vention.

Dynamics of ammonium and pH in the solution of soils with different salinity levels, growing irrigated rice

Rice in Rio Grande do Sul State is grown mostly under flooding, which induces a series of chemical, physical and biological changes in the root environment. These changes, combined with the presence of rice plants, affect the availability of exchangeable ammonium (NH4+) and pH of soil solution, whereas the dynamics of both variables can be influenced by soil salinity, a common problem in the coastal region. This study was conducted to evaluate the dynamics of exchangeable NH4+ and pH in the soil solution, and their relation in the solution of Albaqualf soils with different salinity levels, under rice. Four field experiments were conducted with soils with exchangeable Na percentage (ESP) of 5.6, 9.0, 21.2, and 32.7 %. Prior to flooding, soil solution collectors were installed at depths of 5, 10 and 20 cm. The soil solution was collected weekly, from 7 to 91 days after flooding (DAF), to analyze exchangeable NH4+ and pH in the samples. Plant tissue was sampled 77 DAF, to determine N uptake and estimate the contribution of other N forms to rice nutrition. The content of exchangeable NH4+ decreased over time at all sites and depths, with a more pronounced reduction in soils with lower salinity levels, reaching values close to zero. A possible contribution of non-exchangeable NH4+ forms and N from soil organic matter to rice nutrition was observed. Soil pH decreased with time in soils with ESP 5.6 and 9.0 %, being positively correlated with the decreasing NH4+ levels at these sites.

Time-resolved imaging of the laser forward transfer of liquids

Time-resolved imaging is carried out to study the dynamics of the laser-induced forward transfer of an aqueous solution at different laser fluences. The transfer mechanisms are elucidated, and directly correlated with the material deposited at the analyzed irradiation conditions. It is found that there exists a fluence range in which regular and well-defined droplets are deposited. In this case, laser pulse energy absorption results in the formation of a plasma, which expansion originates a cavitation bubble in the liquid. After the further expansion and collapse of the bubble, a long and uniform jet is developed, which advances at a constant velocity until it reaches the receptor substrate. On the other hand, for lower fluences no material is deposited. In this case, although a jet can be also generated, it recoils before reaching the substrate. For higher fluences, splashing is observed on the receptor substrate due to the bursting of the cavitation bubble. Finally, a discussion of the possible mechanisms which lead to such singular dynamics is also provided.

Some spectral properties of the canonical solution operator to $\bar\partial$ on weighted Fock spaces

We characterize the Schatten class membership of the canonical solution operator to $\overline{\partial}$ acting on $L^2(e^{-2\phi})$, where $\phi$ is a subharmonic function with $\Delta\phi$ a doubling measure. The obtained characterization is in terms of $\Delta\phi$. As part of our approach, we study Hankel operators with anti-analytic symbols acting on the corresponding Fock space of entire functions in $L^2(e^{-2\phi})$

Identification of GPx8 as a novel cellular substrate of the hepatitis C virus NS3-4A protease

Background: The hepatitis C virus (HCV) NS3-4A protease is not only an essential component of the viral replication complex and a prime target for a ntiviral intervention but also a key player i n the persistence and pathogenesis of HCV. It cleaves and thereby inactivates two crucial adaptor proteins in viral RNA sensing and innate immunity (MAVS and TRIF) as well as a phosphatase involved in growth factor signaling (TCPTP). T he aim of this study was to identify novel cellular substrates o f the N S3-4A protease and to investigate their role in the replication and pathogenesis of HCV. Methods: Cell lines inducibly expressing t he NS3-4A protease were analyzed in basal as well as interferon-α-stimulated states by stable isotopic l abeling using amino acids in cell culture (SILAC) coupled with protein separation and mass spectrometry. Candidates fulfilling stringent criteria for potential substrates or products of the NS3-4A protease were further i nvestigated in different experimental systems as well a s in liver biopsies from patients with chronic hepatitis C. Results: SILAC coupled with protein separation and mass spectrometry yielded > 5000 proteins of which 18 candidates were selected for further analyses. These allowed us to identify GPx8, a membrane-associated peroxidase involved in disulfide bond formation in the endoplasmic reticulum, as a n ovel cellular substrate of the H CV NS3-4A protease. Cleavage occurs at cysteine in position 11, removing the cytosolic tip of GPx8, and was observed in different experimental systems as well as in liver biopsies from patients with chronic hepatitis C. Further functional studies, involving overexpression and RNA silencing, revealed that GPx8 is a p roviral factor involved in viral particle production but not in HCV entry or HCV RNA replication. Conclusions: GPx8 is a proviral host factor cleaved by the HCV NS3-4A protease. Studies investigating the consequences of GPx8 cleavage for protein function are underway. The identification of novel cellular substrates o f the HCV N S3-4A protease should yield new insights i nto the HCV life cycle and the pathogenesis of hepatitis C and may reveal novel targets for antiviral intervention.

Quantitative proteomics identifies the membrane-associated peroxidase GPx8 as a cellular substrate of the hepatitis C virus NS3-4A protease.

The hepatitis C virus (HCV) NS3-4A protease is not only an essential component of the viral replication complex and a prime target for antiviral intervention but also a key player in the persistence and pathogenesis of HCV. It cleaves and thereby inactivates two crucial adaptor proteins in viral RNA sensing and innate immunity, mitochondrial antiviral signaling protein (MAVS) and TRIF, a phosphatase involved in growth factor signaling, T-cell protein tyrosine phosphatase (TC-PTP), and the E3 ubiquitin ligase component UV-damaged DNA-binding protein 1 (DDB1). Here we explored quantitative proteomics to identify novel cellular substrates of the NS3-4A protease. Cell lines inducibly expressing the NS3-4A protease were analyzed by stable isotopic labeling using amino acids in cell culture (SILAC) coupled with protein separation and mass spectrometry. This approach identified the membrane-associated peroxidase GPx8 as a bona fide cellular substrate of the HCV NS3-4A protease. Cleavage by NS3-4A occurs at Cys 11, removing the cytosolic tip of GPx8, and was observed in different experimental systems as well as in liver biopsies from patients with chronic HCV. Overexpression and RNA silencing studies revealed that GPx8 is involved in viral particle production but not in HCV entry or RNA replication. Conclusion: We provide proof-of-concept for the use of quantitative proteomics to identify cellular substrates of a viral protease and describe GPx8 as a novel proviral host factor targeted by the HCV NS3-4A protease. (Hepatology 2014;59:423-433).

JNK3 maintains expression of the insulin receptor substrate 2 (IRS2) in insulin-secreting cells: functional consequences for insulin signaling.

We have recently shown that silencing of the brain/islet specific c-Jun N-terminal Kinase3 (JNK3) isoform enhances both basal and cytokine-induced beta-cell apoptosis, whereas silencing of JNK1 or JNK2 has opposite effects. While it is known that JNK1 or JNK2 may promote apoptosis by inhibiting the activity of the pro-survival Akt pathway, the effect of JNK3 on Akt has not been documented. This study aims to determine the involvement of individual JNKs and specifically JNK3 in the regulation of the Akt signaling pathway in insulin-secreting cells. JNK3 silencing strongly decreases Insulin Receptor Substrate 2 (IRS2) protein expression, and blocks Akt2 but not Akt1 activation by insulin, while the silencing of JNK1 or JNK2 activates both Akt1 and Akt2. Concomitantly, the silencing of JNK1 or JNK2, but not of JNK3, potently phosphorylates the glycogen synthase kinase3 (GSK3β). JNK3 silencing also decreases the activity of the transcription factor Forkhead BoxO3A (FoxO3A) that is known to control IRS2 expression, in addition to increasing c-Jun levels that are known to inhibit insulin gene expression. In conclusion, we propose that JNK1/2 on one hand and JNK3 on the other hand, have opposite effects on insulin-signaling in insulin-secreting cells; JNK3 protects beta-cells from apoptosis and dysfunction mainly through maintenance of a normal IRS2 to Akt2 signaling pathway. It seems that JNK3 mediates its effects mainly at the transcriptional level, while JNK1 or JNK2 appear to mediate their pro-apoptotic effect in the cytoplasm.

Use of the Flixene vascular access graft as an early cannulation solution.

OBJECTIVE: The primary end points of this study were safety and efficacy of early cannulation of the Flixene graft (Maquet-Atrium Medical, Hudson, NH). Secondary end points were complications and patency. METHODS: This is a prospective single-center nonrandomized study. Study data included patient characteristics; history of vascular access; operative technique; interval between implantation and initial cannulation; complications; and patency at 1 month, 3 months, and every 6 months. Patency rates were estimated by the Kaplan-Meier method. RESULTS: Between January 2011 and September 2013, a total of 46 Flixene grafts were implanted in 44 patients (27 men) with a mean age of 63 years. The implantation site was the upper arm in 67% of cases, the forearm in 11%, and the thigh in 22%. Seven grafts were never cannulated during the study period. Of the remaining 39 grafts, 32 (82%) were successfully cannulated within the first week after implantation, including 16 (41%) on the first day. The median interval from implantation to initial cannulation was 2 days (interquartile range, 1-3 days). The median follow-up was 223.5 days (interquartile range, 97-600 days). Five hematomas occurred, but only one required surgical revision. Primary assisted and secondary patency rates were 65% and 86%, respectively, at 6 months and 56% and 86%, respectively, at 1 year. CONCLUSIONS: This study suggests that cannulation of the Flixene graft within 1 week after implantation is safe and effective. Early cannulation avoids or shortens the need for a temporary catheter. One-year patency rates appeared to be comparable to those achieved with conventional grafts, but long-term follow-up and randomized controlled studies will be needed to confirm this finding.

Solution equilibria of cytosine- and guanine-rich sequences near the promoter region of the n-myc gene that contain stable hairpins within lateral loops

Cytosine-and guanine-rich regions of DNA are capable of forming complex structures named i-motifs and G-quadruplexes, respectively. In the present study the solution equilibria at nearly physiological conditions of a 34 -bases long cytosine-rich sequence and its complementary guanin e-rich strand corresponding to the first intron of the n-mycgene were studied. Both sequences , not yet studied, contain a 12 - base tract capable of forming stable hairpins inside the i-motif and G-quadruplex structures, respectively ...

Inclusion complex of the antiviral drug acyclovir with cyclodextrin in aqueous solution and in solid phase

Complexation between acyclovir (ACV), an antiviral drug used for the treatment of herpes simplex virus infection, and beta-cyclodextrin (beta-CD) was studied in solution and in solid states. Complexation in solution was evaluated using solubility studies and nuclear magnetic resonance spectroscopy (¹H-NMR). In the solid state, X-ray diffraction, differential scanning calorimetry (DSC), thermal gravimetric analysis (TGA) and dissolution studies were used. Solubility studies suggested the existence of a 1:1 complex between ACV and beta-CD. ¹H-NMR spectroscopy studies showed that the complex formed occurs with a stoichiometry ratio of 1:1. Powder X-ray diffraction indicated that ACV exists in a semicrystalline state in the complexed form with beta-CD. DSC studies showed the existence of a complex of ACV with beta-CD. The TGA studies confirmed the DSC results of the complex. Solubility of ACV in solid complexes was studied by the dissolution method and it was found to be much more soluble than the uncomplexed drug.

Technological solution for the automatic replacement of the catalytic filaments in HWCVD

The degradation of the catalytic filaments is the main factor limiting the industrial implementation of the hot wire chemical vapor deposition (HWCVD) technique. Up to now, no solution has been found to protect the catalytic filaments used in HWCVD without compromising their catalytic activity. Probably, the definitive solution relies on the automatic replacement of the catalytic filaments. In this work, the results of the validation tests of a new apparatus for the automatic replacement of the catalytic filaments are reported. The functionalities of the different parts have been validated using a 0.2 mm diameter tungsten filament under uc-Si:H deposition conditions.