The TOR pathway comes of age

Studies in a variety of model organisms indicate that nutrient signaling is tightly coupled to longevity. In nutrient replete conditions, organisms develop, grow, and age quickly. When nutrients become sparse as with dietary restriction, growth and development decline, stress response pathways become induced and organisms live longer. Considerable effort has been devoted to understanding the molecular events mediating lifespan extension by dietary restriction. One central focus has been on nutrient-responsive signal transduction pathways including insulin/IGF-1, AMP kinase, protein kinase A and the TOR pathway. Here we describe the increasingly prominent links between TOR signaling and aging in invertebrates. Longevity studies in mammals are not published to date. Instead, we highlight studies in mouse models, which indicate that dampening the TOR pathway leads to widespread protection from an array of age-related diseases.     

Stabilized baculovirus vector expressing a heterologous gene and GP64 from a single bicistronic transcript

The efficient scale-up of recombinant protein production in insect-cell bioreactors using baculovirus expression vectors is hampered by reductions in yield with increasing viral passage, the so-called passage effect. This phenomenon is characterized by the generation and subsequent accumulation of defective interfering baculoviruses (DIs), which interfere with the replication of genomically intact virus. A novel baculovirus expression vector is presented equipped with a bicistronic expression cassette that allows the simultaneous expression of the recombinant gene (GFP, first cistron) and an essential baculovirus gene (GP64, second cistron) from a single messenger RNA (mRNA). The translation of GP64 is mediated by an internal ribosome entry site (IRES) element from Rhopalosiphum padi virus (RhPV) while the native GP64 gene is deleted. In this way, a dominant selection pressure is placed on the entire bicistronic mRNA and hence on the maintenance of the foreign gene. The bicistronic expression vector was superior to the control baculovirus vector in that GFP expression remained at much higher levels upon continued virus passage. The versatility of this stabilized vector was demonstrated by its ability to propagate in a number of cell lines including Sf21, Sf9 and High Five cells. This novel baculovirus vector is especially valuable for large-scale recombinant protein production in insect-cell bioreactors where the number of viral passages is high.     

Physical Activity Is not Associated with Estimated Glomerular Filtration Rate among Young and Middle-Aged Adults: Results from the Population-Based Longitudinal Doetinchem Study

There is debate as to whether physical inactivity is associated with reduced kidney function. We studied the prospective association of (changes in) physical activity with estimated glomerular filtration rate (eGFR) in adult men and women. We included 3,935 participants aged 26 to 65 years from the Doetinchem Cohort study, examined every 5 years for 15 years. Physical activity was assessed at each round using the Cambridge Physical Activity Index. Using the CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration) equation, GFR was estimated from routinely measured cystatin C concentrations, examining all available samples per participant in one assay run. We determined the association between 1) physical activity and eGFR and 2) 5-year changes in physical activity (becoming inactive, staying inactive, staying active, becoming active) and eGFR, using time-lagged generalized estimating equation analyses. At baseline, 3.6% of the participants were inactive, 18.5% moderately inactive, 26.0% moderately active, and 51.9% active. The mean (± SD) eGFR was 107.9 (± 14.5) mL/min per 1.73 m². Neither physical activity nor 5-year changes in physical activity were associated with eGFR at the subsequent round. The multivariate adjusted βeGFR was 0.57 mL/min per 1.73 m² (95% Confidence Interval (CI) -1.70, 0.56) for inactive compared to active participants. Studying changes in physical activity between rounds, the adjusted βeGFR was -1.10 mL/min per 1.73 m² (95% CI -4.50, 2.30) for those who stayed inactive compared with participants who became active. Physical activity was not associated with eGFR in this population-based study of adults.     

pH-induced conformational changes in chromatin subunits measured by circular dichroism and immunochemical reactivity

Changes in the conformational state of chromatin core particles from chicken erythrocytes were studied by both immunochemical and biophysical methods as a function of pH and ionic strength. When the pH of core particles in a solution of ionic strength 3, 60 or 220 mM was lowered from pH 7.5, a sharp transition in the circular dichroism spectrum of DNA monitored between 320 and 260 nm was observed at pH 6.65. This change in DNA ellipticity was totally reversible. Binding to core particles of antibodies specific for histones H2B, H2A, H3 and for the IRGERA (synthetic C-terminal) peptide of H3 was used to follow changes in histone antigenicity. Binding was studied in the pH range 7.5-5.35, and at ionic strength of 60 and 220 mM. A change in reactivity of some histone epitopes was observed around pH 6.2–6.5. However, the changes observed by circular dichroism and antibody binding pertain to different components of chromatin subunits and they probably reflect independent phenomena. The alteration in accessibility of these determinants at the surface of core particles was completely reversible and was dependent on ionic strength. The conformation changes in core particles occurring near physiological ionic strength and pH may reflect dynamic changes in chromatin structure that possess functional significance.     

Novel small renin inhibitors containing 4,5- or 3,5-dihydroxy-2-substituted-6-phenylhexanamide replacements at the P2P3 sites

Renin inhibitors containing a 4,5- or a 3,5-dihydroxy-2-substituted-6-phenylhexanamide fragment at the P₂---P₃ sites have been prepared and evaluated. The four possible diastereomeric diols of the two series of inhibitors were synthesized to determine the optimal configuration of the carbinol centers for these replacements. The most potent inhibitors of each series, 1a and 2c have a molecular weight of only 503 and IC₅₀ values of 23 and 20 nM in a human plasma renin assay at pH 6.0. Their very low aqueous solubility limited their further evaluation. The efficacy of these P₂---P₃ replacements is a result of their ability to maintain the important hydrogen-bonds with the enzyme. Due to conformational differences with the dipeptide, adjustment at the P₂ side chain was required. These 4,5- and 3,5-dihydroxyhexanamide segments could be seen as novel N-terminal dipeptide replacements.     

Freeze-fracture study of ordered arrays of particles in the plasma membrane ofChlamydobotrys stellata Korsch. (Volvocales)

Summary Ultrarapid cyrofixation procedures revealed the existence of ordered arrays of intramembrane particles on E fracture faces and corresponding ordered imprints on P faces in freeze-fractured plasma membrane of the green algaeChlamydobotrys stellata (Korschikoff). The structure of these arrays is very sensitive to cryofixation conditions and particularly to glutaraldehyde prefixation which leads to the formation of amorphous two-dimensional aggregates. The size of the individual ordered arrays and the ratio of ordered to total surface of the membrane increase with growth temperature from 15°C to 30°C with a corresponding decrease in cell generation time. Above 30°C the size of the individual ordered arrays decreases. At high, but sublethal temperature (above 37°C) the ordered arrays become smaller. In addition to the predominant two-dimensional oblique organization (a=12.0nm, b=12.6nm, =80°), square and tetragonal arrangements are also present. The cell wall is composed of many layers, one of which displays a zipper-like structure composed of periodic ridges 25 nm distant, sandwiched between two more or less fibrillar layers. The appearance and changes of the organization of ordered arrays are discussed in relation to their eventual physiological role during the life cycle of the cells and in particular to the formation of the cell wall and the median periodic leaflet.Dedicated to our late colleague and friend Dr.Yvonne Henry.     

Modeling anaerobic digestion of microalgae using ADM1

The coupling between a microalgal pond and an anaerobic digester is a promising alternative for sustainable energy production by transforming carbon dioxide into methane using solar energy. In this paper, we demonstrate the ability of the original ADM1 model and a modified version (based on Contois kinetics for the hydrolysis steps) to represent microalgae anaerobic digestion. Simulations were compared to experimental data of an anaerobic digester fed with Chlorella vulgaris. The modified ADM1 fits adequately the data for the considered 140 day experiment encompassing a variety of influent load and flow rates. It turns out to be a reliable predictive tool for optimising the coupling of microalgae with anaerobic digestion processes.     

Comparison of the Performance of Cartomizer Style Electronic Cigarettes from Major Tobacco and Independent Manufacturers

Objective

This study compared the performance of 12 brands of cartomizer style electronic cigarettes (EC) using different puffing protocols and measured the concentrations of nicotine in each product.

Methods

Air flow rate, pressure drop, and aerosol absorbance were measured using two different protocols, first 10 puffs and a modified smoke-out protocol.

Results

First 10 puff protocol: The air flow rate required to produce aerosol ranged between brands from 4–21 mL/s. Pressure drop was relatively stable within a brand but ranged between brands from 14–71 mmH₂O and was much lower than the earlier classic 3-piece models. Absorbance, a measure of aerosol density, was relatively consistent between puffs, but varied between brands. With the modified smoke-out protocol, most brands were puffed until 300 puffs. The pressure drop was relatively stable for all brands except three. Absorbance of the aerosol decreased as the number of puffs increased. Although there was some uniformity in performance within some brands, there was large variation between brands. The labeled and measured nicotine concentrations were within 10% of each other in only 1 out of 10 brands.

Conclusions

Over 10 puffs, the cartomizers all perform similarly within a brand but varied between brands. In smoke-out trials, most brands lasted at least 300 puffs, and performed similarly within brands with respect to pressure drop and absorbance. For five brands, products purchased at different times performed differently. These data show some improvement in performance during evolution of these products, but nevertheless indicate problems with quality control in manufacture.