Understanding the α-crystallin cell membrane conjunction

PURPOSE. It is well established that levels of soluble α-crystallin in the lens cytoplasm fall steadily with age, accompanied by a corresponding increase in the amount of membrane-bound α-crystallin. Less well understood, is the mechanism driving this age-dependent membrane association. The aim of this study was to investigate the role of the membrane and its associated proteins and peptides in the binding of α-crystallin. METHODS. Fibre cell membranes from human and bovine lenses were separated from soluble proteins by centrifugation. Membranes were stripped of associated proteins with successive aqueous, urea and alkaline solutions. Protein constituents of the respective membrane isolates were examined by SDS-PAGE and Western immunoblotting. Recombinant αA- and αB-crystallins were fluorescently-labeled with Alexa350® dye and incubated with the membrane isolates and the binding capacity of membrane for α-crystallin was determined. RESULTS. The binding capacity of human membranes was consistently higher than that of bovine membranes. Urea- and alkali-treated membranes from the nucleus had similar binding capacities for αA-crystallin, which were significantly higher than both cortical membrane extracts. αB-Crystallin also had a higher affinity for nuclear membrane. However, urea-treated nuclear membrane had three times the binding capacity for αB-crystallin as compared to the alkali-treated nuclear membrane. Modulation of the membrane-crystallin interaction was achieved by the inclusion of an N-terminal peptide of αB-crystallin in the assays, which significantly increased the binding. Remarkably, following extraction with alkali, full length αA- and αB-crystallins were found to remain associated with both bovine and human lens membranes. CONCLUSIONS. Fiber cell membrane isolated from the lens has an inherent capacity to bind α-crystallin. For αB-crystallin, this binding was found to be proportional to the level of extrinsic membrane proteins in cells isolated from the lens nucleus, indicating these proteins may play a role in the recruitment of αB-crystallin. No such relationship was evident for αA-crystallin in the nucleus, or for cortical membrane binding. Intrinsic lens peptides, which increase in abundance with age, may also function to modulate the interaction between soluble α-crystallin and the membrane. In addition, the tight association between α-crystallin and the lens membrane suggests that the protein may be an intrinsic component of the membrane structure

Long-term Periodicities of Cataclysmic Variables with Synoptic Surveys

A systematic study on the long-term periodicities of known Galactic cataclysmic variables (CVs) was conducted. Among 1580 known CVs, 344 sources were matched and extracted from the Palomar Transient Factory (PTF) data repository. The PTF light curves were combined with the Catalina Real-Time Transient Survey (CRTS) light curves and analyzed. Ten targets were found to exhibit long-term periodic variability, which is not frequently observed in the CV systems. These long-term variations are possibly caused by various mechanisms, such as the precession of the accretion disk, hierarchical triple star system, magnetic field change of the companion star, and other possible mechanisms. We discuss the possible mechanisms in this study. If the long-term period is less than several tens of days, the disk precession period scenario is favored. However, the hierarchical triple star system or the variations in magnetic field strengths are most likely the predominant mechanisms for longer periods.Comment: 33 pages, 9 figures (manuscript form), Accepted for publication in PAS

Safety and efficacy of ceftolozane/tazobactam plus metronidazole versus meropenem from a phase 2, randomized clinical trial in pediatric participants with complicated intra-abdominal infection

BACKGROUND: Ceftolozane/tazobactam, a cephalosporin-β-lactamase inhibitor combination, is approved for the treatment of complicated urinary tract infections and complicated intra-abdominal infections (cIAI). The safety and efficacy of ceftolozane/tazobactam in pediatric participants with cIAI were assessed. METHODS: This phase 2 study (NCT03217136) randomized participants to either ceftolozane/tazobactam+metronidazole or meropenem for treatment of cIAI in pediatric participants (\u3c18 years). The primary objective was to assess the safety and tolerability of intravenous ceftolozane/tazobactam+metronidazole. Clinical cure at end of treatment (EOT) and test of cure (TOC) visits were secondary end points. RESULTS: The modified intent-to-treat (MITT) population included 91 participants (ceftolozane/tazobactam+metronidazole, n = 70; meropenem, n = 21). Complicated appendicitis was the most common diagnosis (93.4%); Escherichia coli was the most common pathogen (65.9%). Adverse events (AEs) occurred in 80.0% and 61.9% of participants receiving ceftolozane/tazobactam+metronidazole and meropenem, drug-related AEs occurred in 18.6% and 14.3% and serious AEs occurred in 11.4% and 0% of participants receiving ceftolozane/tazobactam+metronidazole and meropenem, respectively. No drug-related serious AEs or discontinuations due to drug-related AEs occurred. Rates of the clinical cure for ceftolozane/tazobactam+metronidazole and meropenem at EOT were 80.0% and 95.2% (difference: -14.3; 95% confidence interval: -26.67 to 4.93) and at TOC were 80.0% and 100.0% (difference: -19.1; 95% confidence interval: -30.18 to -2.89), respectively; 6 of the 14 clinical failures for ceftolozane/tazobactam+metronidazole at TOC were indeterminate responses imputed as failures per protocol. CONCLUSION: Ceftolozane/tazobactam+metronidazole was well tolerated in pediatric participants with cIAI and had a safety profile similar to the established safety profile in adults. In this descriptive efficacy analysis, ceftolozane/tazobactam+metronidazole appeared efficacious

The Ino80 complex prevents invasion of euchromatin into silent chromatin

Here we show that the Ino80 chromatin remodeling complex (Ino80C) directly prevents euchromatin from invading transcriptionally silent chromatin within intergenic regions and at the border of euchromatin and heterochromatin. Deletion of Ino80C subunits leads to increased H3K79 methylation and noncoding RNA polymerase II (Pol II) transcription centered at the Ino80C-binding sites. The effect of Ino80C is direct, as it blocks H3K79 methylation by Dot1 in vitro. Heterochromatin stimulates the binding of Ino80C in vitro and in vivo. Our data reveal that Ino80C serves as a general silencing complex that restricts transcription to gene units in euchromatin

Elucidation of Binding Determinants and Functional Consequences of Ras/Raf-Cysteine-rich Domain Interactions

Raf-1 is a critical downstream target of Ras and contains two distinct domains that bind Ras. The first Ras-binding site (RBS1) in Raf-1 has been shown to be essential for Ras-mediated translocation of Raf-1 to the plasma membrane, whereas the second site, in the Raf-1 cysteine-rich domain (Raf-CRD), has been implicated in regulating Raf kinase activity. While recognition elements that promote Ras.RBS1 complex formation have been characterized, relatively little is known about Ras/Raf-CRD interactions. In this study, we have characterized interactions important for Ras binding to the Raf-CRD. Reconciling conflicting reports, we found that these interactions are essentially independent of the guanine nucleotide bound state, but instead, are enhanced by post-translational modification of Ras. Specifically, our findings indicate that Ras farnesylation is sufficient for stable association of Ras with the Raf-CRD. Furthermore, we have also identified a Raf-CRD variant that is impaired specifically in its interactions with Ras. NMR data also suggests that residues proximal to this mutation site on the Raf-CRD form contacts with Ras. This Raf-CRD mutant impairs the ability of Ras to activate Raf kinase, thereby providing additional support that Ras interactions with the Raf-CRD are important for Ras-mediated activation of Raf-1

Effect of food matrix and processing on release of almond protein during simulated digestion

Abstract The aims of the present work were to assess digestibility of almond protein in the upper gastrointestinal tract, evaluate the effects of food matrix on protein release and assess the persistence of immunoreactive polypeptides generated during simulated digestion. Prunin, the most abundant protein in almond flour, was sensitive to pepsin, with complete digestion after 20 min in the gastric phase. Addition of the surfactant phosphatidylcholine did not affect the rate and kinetic of digestion, as observed by SDS-PAGE analysis and HPLC, in the stomach and the small intestine of either natural or blanched almond flour. However, incorporation of almond flour into a food matrix, such as chocolate mousse and Victorian sponge cake, decreased the rate of almond protein degradation by pepsin and immunoreactivity of almond polypeptides detected by dot blots and sandwich ELISA retained better. Most of the almond protein identified by in-gel tryptic digestion and MALDI-TOF analysis corresponded to prunin, with pI values of 5–7. Further human sera studies are warranted to investigate the relationship between food matrix and almond allergy

The Antiferromagnetic Band Structure of La2CuO4 Revisited

Using the Becke-3-LYP functional, we have performed band structure calculations on the high temperature superconductor parent compound, La2CuO4. Under the restricted spin formalism (rho(alpha) equal to rho(beta)), the R-B3LYP band structure agrees well with the standard LDA band structure. It is metallic with a single Cu x2-y2/O p(sigma) band crossing the Fermi level. Under the unrestricted spin formalism (rho(alpha) not equal to rho(beta)), the UB3LYP band structure has a spin polarized antiferromagnetic solution with a band gap of 2.0 eV, agreeing well with experiment. This state is 1.0 eV (per formula unit) lower than that calculated from the R-B3LYP. The apparent high energy of the spin restricted state is attributed to an overestimate of on-site Coulomb repulsion which is corrected in the unrestricted spin calculations. The stabilization of the total energy with spin polarization arises primarily from the stabilization of the x2-y2 band, such that the character of the eigenstates at the top of the valence band in the antiferromagnetic state becomes a strong mixture of Cu x2-y2/O p(sigma) and Cu z2/O' p(z). Since the Hohenberg-Kohn theorem requires the spin restricted and spin unrestricted calculations give exactly the same ground state energy and total density for the exact functionals, this large disparity in energy reflects the inadequacy of current functionals for describing the cuprates. This calls into question the use of band structures based on current restricted spin density functionals (including LDA) as a basis for single band theories of superconductivity in these materials.Comment: 13 pages, 8 figures, to appear in Phys. Rev. B, for more information see http://www.firstprinciples.co

Economic evaluation of the specialized donor care facility for thoracic organ donor management

Background: Over the last decade two alternative models of donor care have emerged in the United States: the conventional model, whereby donors are managed at the hospital where brain death occurs, and the specialized donor care facility (SDCF), in which brain dead donors are transferred to a SDCF for medical optimization and organ procurement. Despite increasing use of the SDCF model, its cost-effectiveness in comparison to the conventional model remains unknown. Methods: We performed an economic evaluation of the SDCF and conventional model of donor care from the perspective of U.S. transplant centers over a 2-year study period. In this analysis, we utilized nationwide data from the Scientific Registry of Transplant Recipients and controlled for donor characteristics and patterns of organ sharing across the nation\u27s organ procurement organizations (OPOs). Subgroup analysis was performed to determine the impact of the SDCF model on thoracic organ transplants. Results: A total of 38,944 organ transplants were performed in the U.S. during the study period from 13,539 donors with an observed total organ cost of $1.36 billion. If every OPO assumed the cost and effectiveness of the SDCF model, a predicted 39,155 organ transplants (+211) would have been performed with a predicted total organ cost of$1.26 billion (-$100 million). Subgroup analysis of thoracic organs revealed that the SDCF model would lead to a predicted 156 additional transplants with a cost saving of$24.6 million. Conclusions: The U.S. SDCF model may be a less costly and more effective means of multi-organ donor management, particularly for thoracic organ donors, compared to the conventional hospital-based model

In Vivo Comparison of Two Human Norovirus Surrogates for Testing Ethanol-Based Handrubs: The Mouse Chasing the Cat!

Human noroviruses (HuNoV), a major cause of acute gastroenteritis worldwide, cannot be readily cultured in the lab. Therefore, a feline calicivirus (FCV) is often used as its surrogate to, among other things, test alcohol-based handrubs (ABHR). The more recent laboratory culture of a mouse norovirus (MNV) provides an alternative. While MNV is closer to HuNoV in several respects, to date, no comparative testing of FCV and MNV survival and inactivation on human hands has been performed. This study was designed to address the knowledge gap. The rates of loss in viability during drying on hands were −1.91 and −1.65% per minute for FCV and MNV, respectively. When the contaminated skin was exposed for 20 s to either a commercial ABHR with 62% (v/v) ethanol or to 75% (v/v) ethanol in water, FCV infectivity was reduced by <1 log10 while that of MNV by nearly 2.8 log10. Extending the contact time to 30 s reduced the FCV titer by almost 2 log10 by both test substances and that of MNV by >3.5 log10 by the commercial ABHR while 75% ethanol did not show any noticeable improvement in activity as compared to the 20 s contact. An 80% (v/v) aqueous solution of ethanol gave only a 1.75 log10 reduction in MNV activity after 20 s. The results show significant differences in the ethanol susceptibility of FCV and MNV in contact times relevant to field use of ABHR and also that 62% ethanol was a more effective virucide than either 75% or 80% ethanol. These findings indicate the need for a review of the continuing use of FCV as a surrogate for HuNoV