A Novel Mechanism for NF-κB-activation via IκB-aggregation: Implications for Hepatic Mallory-Denk-Body Induced Inflammation

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Highlights

• •Liver Mallory-Denk-Body inducers elicited an IκBα-loss and NF-κB-activation.
• •IκBα-loss was due to its sequestration into insoluble cytoplasmic aggregates.
• •Four proteomic approaches identified 10 IκBα-interacting/aggregating proteins.
• •Nup153/RanBP2-aggregation prevented IκBα nuclear entry for ending NF-κB-activation.

     

The activity of myofibrillar and actomyosin ATPase in the skeletal muscle of some marine teleosts in relation to their length and age

The activity of myofibrillar ATPase declined with length in the five species examined ( Merlanogrummus aeglefinis L., Merrluccius merluccius L., Physis blennoides Brunnich, Pleuronectes platessa L. and Merlangius merlangus L.). This decline was related to a decrease in the in vitro muscle contraction rate and appeared to be a scale rather than an age effect. Actomyosin was extracted from Merlangius merlangus L. to investigate the length related changes in the myofibrillar protein.     

Coupling of M3 Acetylcholine Receptor to Gq16 Activates a Natriuretic Peptide Receptor Guanylyl Cyclase

Muscarinic activation of tracheal smooth muscle (TSM) involves a M₃AChR/heterotrimeric-G protein/NPR-GC coupling mechanism. G protein activators Mastoparan (MAS) and Mastoparan-7 stimulated 4- and 10-fold the NPR-GC respectively, being insensitive to PTX and antibodies against Gα_i/o subfamily. Muscarinic and MAS stimulation of NPR-GC was blocked by antibodies against C-terminal of Gα_q16, whose expression was confirmed by RT-PCR. However, synthetic peptides from C-terminal of Gα_q15/16 stimulated the NPR-GC. Coupling of α_q16 to M₃AChR is supported by MAS decreased [³H]QNB binding, being abolished after M₃AChR-4-DAMP-alkylation. Anti-i₃M₃AChR antibodies blocked the muscarinic activation of NPR-GC, and synthetic peptide from i₃M₃AChR (M₃P) was more potent than MAS increasing GTPγ [³⁵S] and decreasing the [³H]QNB activities. Coupling between NPR-GC and Gα_q16 was evaluated by using trypsin-solubilized-fraction from TSM membranes, which displayed a MAS-sensitive-NPR-GC activity, being immunoprecipitated with anti-Gα_q16, also showing an immunoreactive heterotrimeric-G-β -subunit. These data support the existence of a novel transducing cascade, involving Gα_q16β γ coupling M₃AChR to NPR-GC.     

Some 1,2-Diphenylethane Derivatives as Inhibitors of Retinoic Acid—Metabolising Enzymes

In a search for novel inhibitors of RA-metabolising enzyme inhibitors as potential anti-cancer agents some 1,2-ethandiones, 2-hydroxyethanones and 1-ethylenedioxyethanones based on aryl-substituted 1,2-diphenylethane have been examined. Several of the compounds were weak inhibitors of the non-specific rat liver microsomal P450 enzymes and moderate inhibitors of the RA-induced enzymes in cultured human genital fibroblasts, where the RA-specific enzyme CYP26 is probably expressed. The 2-hydroxyethanone (13) with a 1-(4-dimethylaminophenyl) substituent was overall the most potent compound for rat liver microsomal enzyme (IC50=52.1?μM; ketoconazole, 2.8?μM) and the RA-induced enzyme (100?μM, 65.9% inhibition; ketoconazole, 20?μM, 75.0%). Modification of the dimethylamino group in (13) with more hydrophobic dialkylamino functions or separate modification of the 2-(2,4-dichlorophenyl) function did not improve potency.     

An IDEA for Short Term Outbreak Projection: Nearcasting Using the Basic Reproduction Number

Background

Communicable disease outbreaks of novel or existing pathogens threaten human health around the globe. It would be desirable to rapidly characterize such outbreaks and develop accurate projections of their duration and cumulative size even when limited preliminary data are available. Here we develop a mathematical model to aid public health authorities in tracking the expansion and contraction of outbreaks with explicit representation of factors (other than population immunity) that may slow epidemic growth.

Methodology

The Incidence Decay and Exponential Adjustment (IDEA) model is a parsimonious function that uses the basic reproduction number R₀, along with a discounting factor to project the growth of outbreaks using only basic epidemiological information (e.g., daily incidence counts).

Principal Findings

Compared to simulated data, IDEA provides highly accurate estimates of total size and duration for a given outbreak when R₀ is low or moderate, and also identifies turning points or new waves. When tested with an outbreak of pandemic influenza A (H1N1), the model generates estimated incidence at the i+1^th serial interval using data from the i^th serial interval within an average of 20% of actual incidence.

Conclusions and Significance

This model for communicable disease outbreaks provides rapid assessments of outbreak growth and public health interventions. Further evaluation in the context of real-world outbreaks will establish the utility of IDEA as a tool for front-line epidemiologists.     

Media supplementation for targeted manipulation of monoclonal antibody galactosylation and fucosylation

Monoclonal antibodies are critically important biologics as the largest class of molecules used to treat cancers, rheumatoid arthritis, and other chronic diseases. Antibody glycosylation is a critical quality attribute that has ramifications for patient safety and physiological efficacy—one that can be modified by such factors as media formulation and process conditions during production. Using a design-of-experiments approach, we examined the effect of 2-F-peracetyl fucose (2FP), uridine, and galactose on cell growth and metabolism, titer, and gene expression of key glycosylation-related proteins, and report how the glycoform distribution changed from Days 4 to 7 in a batch process used for IgG1 production from Chinese hamster ovary cells. We observed major glycosylation changes upon supplement addition, where the addition of 2FP decreased antibody fucosylation by up to 48%, galactose addition increased galactosylation by up to 21%, and uridine addition decreased fucosylation and increased galactosylation by 6% and 2%, respectively. Despite having major effects on glycosylation, neither galactose nor 2FP significantly affected cell culture growth, metabolism, or titer. Uridine improved peak cell densities by 23% but also reduced titer by ∼30%. The supplements caused significant changes in gene expression by Day 4 of the cultures where 2FP addition significantly reduced fucosyltransferase 8 and nucleotide sugar transporter gene expression (by ∼2-fold), and uridine addition significantly increased expression of UDP-GlcNAcT (SLC35A3) and B4GALT1–6 genes (by 1.5–3-fold). These gene expression data alongside glycosylation, metabolic, and growth data improve our understanding of the cellular mechanisms affected by media supplementation and suggest approaches for modifying antibody glycosylation in antibody production processes.     

Calpain activation mediates microgravity-induced myocardial abnormalities in mice via p38 and ERK1/2 MAPK pathways

The human cardiovascular system has adapted to function optimally in Earth''s 1G gravity, and microgravity conditions cause myocardial abnormalities, including atrophy and dysfunction. However, the underlying mechanisms linking microgravity and cardiac anomalies are incompletely understood. In this study, we investigated whether and how calpain activation promotes myocardial abnormalities under simulated microgravity conditions. Simulated microgravity was induced by tail suspension in mice with cardiomyocyte-specific deletion of Capns1, which disrupts activity and stability of calpain-1 and calpain-2, and their WT littermates. Tail suspension time-dependently reduced cardiomyocyte size, heart weight, and myocardial function in WT mice, and these changes were accompanied by calpain activation, NADPH oxidase activation, and oxidative stress in heart tissues. The effects of tail suspension were attenuated by deletion of Capns1. Notably, the protective effects of Capns1 deletion were associated with the prevention of phosphorylation of Ser-345 on p47^phox and attenuation of ERK1/2 and p38 activation in hearts of tail-suspended mice. Using a rotary cell culture system, we simulated microgravity in cultured neonatal mouse cardiomyocytes and observed decreased total protein/DNA ratio and induced calpain activation, phosphorylation of Ser-345 on p47^phox, and activation of ERK1/2 and p38, all of which were prevented by calpain inhibitor-III. Furthermore, inhibition of ERK1/2 or p38 attenuated phosphorylation of Ser-345 on p47^phox in cardiomyocytes under simulated microgravity. This study demonstrates for the first time that calpain promotes NADPH oxidase activation and myocardial abnormalities under microgravity by facilitating p47^phox phosphorylation via ERK1/2 and p38 pathways. Thus, calpain inhibition may be an effective therapeutic approach to reduce microgravity-induced myocardial abnormalities.     

Individual and Coupled Effects of Echinoderm Extracts and Surface Hydrophobicity on Spore Settlement and Germination in the Brown Alga Hincksia irregularis

Marine substrata possess cues that influence the behavior of fouling organisms. Initial adhesion of fouling algal zoospores to surfaces is also theorized to depend primarily upon interactions between substrata and spore cell bodies and flagellar membranes. In an effort to identify cues and surface characteristics that influence spore settlement and early development, the effects of bioactive echinoderm extracts, surface charge, and surface hydrophobicity were examined individually and in tandem on zoospore settlement and germination in Hincksia irregularis. Experiments utilizing 96-well plastic culture plates confirmed that spore settlement and germination were significantly affected by surface charge and hydrophobicity as well as by echinoderm metabolites, both individually and in tandem. Spore settlement rates in the dark over 30?min were >?400% higher on hydrophobic surfaces than on positively and negatively charged surfaces. Spore germling numbers were >?300% higher on hydrophobic surfaces than on positively and negatively charged surfaces when spores were allowed to settle in the light for 30?min and the settled spores allowed to subsequently germinate for 24?h. Spore germling numbers were consistently >?25% higher on hydrophobic surfaces than on positively and negatively charged surfaces when equal numbers of spores were allowed to completely settle in the light and subsequently germinate for 24?h. H. irregularis germ tube lengths were also significantly longer on positively charged plates than on negatively charged plates. All echinoderm extracts tested had significant effects on germination and settlement at levels below those of estimated ecological concentrations. Short-term (30?min) exposure and subsequent germination experiments indicated that higher concentrations of extracts had rapid toxic effects on algal spores. Synchronous effects of echinoderm extracts and plate charge upon spore settlement varied considerably and did not show a strong dose response relationship. Long-term (24?h) exposure of spores to echinoderm extracts had dosage dependent effects on germination and spore survival. The results of this study indicate that H. irregularis spores possess the capacity for complex responses to their environment, utilizing combined cues of surface charge, surface energy and biochemistry to determine where to settle and germinate. These responses may aid spores in the detection of suitable substrata and conditions for settlement in the marine environment.