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Efavirenz (EFV) is a non-nucleoside reverse transcriptase inhibitor (NNRTI) widely used in human immunodeficiency virus (HIV) infection therapy. It has been associated with hepatotoxic effects and alterations in lipid and body fat composition. Given the importance of the liver in lipid regulation, we have evaluated the effects of clinically used concentrations of EFV on the mitochondria and lipid metabolism of human hepatic cells in vitro. Mitochondrial function was rapidly undermined by EFV to an extent that varied with the concentration employed; in particular, respiration and intracellular adenosine triphosphate (ATP) levels were reduced whereas reactive oxygen species (ROS) production increased. Results in isolated mitochondria suggest that the mechanism responsible for these actions was a specific inhibition of complex I of the respiratory chain. The reduction in energy production triggered a compensatory mechanism mediated by the enzyme adenosine monophosphate-activated protein kinase (AMPK), the master switch of cellular bioenergetics. Fluorescence and nuclear magnetic resonance demonstrated a rapid intracellular increase of neutral lipids, usually in the form of droplets. This was prevented by the AMPK inhibitor compound C and by removal of fatty acids from the culture medium. These effects were not reproduced by Nevirapine, another NNRTI. EFV is clinically coadministered with two nucleoside reverse transcriptase inhibitors. Evaluation of one of the most common combination, EFV/Lamivudine/Abacavir, revealed that the effects of EFV on ROS production were enhanced. Conclusion: Clinical concentrations of EFV induce bioenergetic stress in hepatic cells by acutely inhibiting mitochondrial function. This new mechanism of mitochondrial interference leads to an accumulation of lipids in the cytoplasm that is mediated by activation of AMPK. (HEPATOLOGY 2010;52:115-125) C ontinuous administration of the drugs included under the term highly active antiretroviral therapy has made acquired immune deficiency syndrome a chronic rather than terminal illness. The initial development of these drugs was particularly rapid and focused on clinical efficacy-reduction of mortality-before all other considerations. However, as the disease has come under control, there has been a growing emphasis on the long-term adverse effects induced by this therapy. Efavirenz (EFV) is a non-nucleoside reverse transcriptase inhibitor (NNRTI) widely used in initial therapy for human immunodeficiency virus (HIV) infection. It is administered to adults in a single daily dose of 600 mg, which Abbreviations: 3TC, lamivudine; ABC, abacavir; AMPK, adenosine monophosphate-activated protein kinase; ATP, adenosine triphosphate; DCFH-DA, 2 0 ,7 0 -dichlorodihydrofluorescein diacetate; EFV, Efavirenz; HIV, human immunodeficiency virus; HR-MAS, high resolution magic angle spectroscopy; NNRTIs, nonnucleoside reverse transcriptase inhibitors; NRTI, nucleoside reverse transcriptase inhibitors; NVP, nevirapine; P-AMPK, phosphorylated adenosine monophosphate...

). Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture. USDA is an equal opportunity provider and employer. SUMMARYSlow movement of molecules in glassy matrices controls the kinetics of chemical and physical reactions in dry seeds. Variation in physiological activity among seeds suggests that there are differences in mobility among seed glasses. Testing this hypothesis is difficult because few tools are available to measure molecular mobility within dry seeds. Here, motional properties within dry pea cotyledons were assessed using dynamic mechanical analysis. The technique detected several molecular relaxations between )80 and +80°C and gave a more detailed description of water content-temperature effects on molecular motion than previously understood from studies of glass formation in seeds at glass transition (Tg). Diffusive movement is delimited by the a relaxation, which appears to be analogous to Tg. b and c relaxations were also detected at temperatures lower than a relaxations, clearly demonstrating intramolecular motion within the glassy matrix of the pea cotyledon. Glass transitions, or the mechanical counterpart a relaxation, appear to be less relevant to seed aging during dry storage than previously thought. On the other hand, b relaxation occurs at temperature and moisture conditions typically used for seed storage and has established importance for physical aging of synthetic polymer glasses. Our data show that the nature and extent of molecular motion varies considerably with moisture and temperature, and that the hydrated conditions used for accelerated aging experiments and ultra-dry conditions sometimes recommended for seed storage give greater molecular mobility than more standard seed storage practices. We believe characterization of molecular mobility is critical for evaluating how dry seeds respond to the environment and persist through time.

Desiccation-tolerant (DT) plant germplasm (i.e. seeds, pollen and spores) survive drying to low moisture contents, when cytoplasm solidifies, forming a glass, and chemical reactions are slowed. DT germplasm may survive for long periods in this state, though inter-specific and intra-specific variation occurs and is not currently explained. Such variability has consequences for agriculture, forestry and biodiversity conservation. Longevity was previously considered in the context of morphological features, cellular constituents or habitat characteristics. We suggest, however, that a biophysical perspective, which considers the molecular organization – or structure – within dried cytoplasm, can provide a more integrated understanding of the fundamental mechanisms that control ageing rates, hence the variation of longevity among species and cell types. Based on biochemical composition and physical–chemical properties of dried materials, we explore three types of the interplay between structural conformations of dried cytoplasm and ageing: (1) cells that lack chlorophyll and contain few storage lipids may exhibit long shelf life, with ageing probably occurring through slow autoxidative processes within the glassy matrix as it relaxes; (2) cells with active chlorophyll may die quickly, possibly because they are prone to oxidative stress promoted by the photosynthetic pigments in the absence of metabolic water and (3) cells that lack chloroplasts but contain high storage lipids may die quickly during storage at −20°C, possibly because lipids crystallize and destabilize the glassy matrix. Understanding the complex variation in structural conformation in space and time may help to design strategies that increase longevity in germplasm with generally poor shelf life.

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