The understanding of the physiological and pathophysiological role of PLTP has greatly increased since the discovery of PLTP more than a quarter of century ago. A comprehensive review of PLTP is presented on the following topics: PLTP gene organization and structure; PLTP transfer properties; different forms of PLTP; characteristics of plasma PLTP complexes; relationship of plasma PLTP activity, mass and specific activity with lipoprotein and metabolic factors; role of PLTP in lipoprotein metabolism; PLTP and reverse cholesterol transport; insights from studies of PLTP variants; insights of PLTP from animal studies; PLTP and atherosclerosis; PLTP and signal transduction; PLTP in the brain; and PLTP in human disease.
PLTP's central role in lipoprotein metabolism and lipid transport in the vascular compartment has been firmly established. However, more studies are needed to further delineate PLTP's functions in specific tissues, such as the lung, brain and adipose tissue. Furthermore, the specific role that PLTP plays in human diseases, such as atherosclerosis, cancer, or neurodegenerative disease, remains to be clarified. Exciting directions for future research include evaluation of PLTP's physiological relevance in intracellular lipid metabolism and signal transduction, which undoubtedly will advance our knowledge of PLTP functions in health and disease.
phospholipid transfer protein; PLTP gene organization and structure; PLTP transfer properties; forms of PLTP; plasma PLTP complexes; lipoprotein metabolism; reverse cholesterol transport; cholesterol efflux; PLTP variants; PLTP animal studies; PLTP and atherosclerosis; PLTP in signal transduction; PLTP in the brain; PLTP in human diseases
Histone acetyltransferase 1 (HAT1) is an enzyme that is likely to be responsible for the acetylation that occurs on lysines 5 and 12 of the NH2-terminal tail of newly synthesized histone H4. Initial studies suggested that, despite its evolutionary conservation, this modification of new histone H4 played only a minor role in chromatin assembly. However, a number of recent studies have brought into focus the important role of both this modification and HAT1 in histone dynamics. Surprisingly, the function of HAT1 in chromatin assembly may extend beyond just its catalytic activity to include its role as a major histone binding protein. These results are incorporated into a model for the function of HAT1 in histone deposition and chromatin assembly.
Transferrin (Tf) is a paradigmatic metalloprotein, which has been extensively studied in the past and still is a focal point of numerous investigation efforts owing to its unique role in iron homeostasis and enormous promise as a component of a wide range of therapies.
SCOPE OF REVIEW
Electrospray ionization mass spectrometry (ESI MS) is a potent analytical tool that has been used successfully to study various properties of Tf and Tf-based products, ranging from covalent structure and metal binding to conformation and interaction with their physiological partners.
Various ESI MS-based techniques produce unique information on Tf properties and behavior that is highly complementary to information provided by other experimental techniques.
The experimental ESI MS-based techniques developed for Tf studies are not only useful for understanding of fundamental aspects of the iron-binding properties of this protein and optimizing Tf-based therapeutic products, but can also be applied to study a range of other metalloproteins.
Neuroimaging biomarkers that precede cognitive decline have the potential to aid early diagnosis of Alzheimer's disease (AD). A body of diffusion tensor imaging (DTI) work has demonstrated declines in white matter (WM) microstructure in AD and its typical prodromal state, amnestic mild cognitive impairment. The present review summarizes recent evidence suggesting that WM integrity declines are present in individuals at high AD-risk, prior to cognitive decline. The available data suggest that AD-risk is associated with WM integrity declines in a subset of tracts showing decline in symptomatic AD. Specifically, AD-risk has been associated with WM integrity declines in tracts that connect grey matter structures associated with memory function. These tracts include parahippocampal WM, the cinglum, the inferior fronto-occipital fasciculus, and the splenium of the corpus callosum. Preliminary evidence suggests that some AD-risk declines are characterized by increases of radial diffusivity, raising the possibility that a myelin-related pathology may contribute to AD onset. These findings justify future research aimed at a more complete understanding of the neurobiological bases of DTI-based declines in AD. With continued refinement of imaging methods, DTI holds promise as a method to aid identification of presymptomatic AD.
DTI; diffusion tensor imaging; APOE; presymptomatic; Alzheimer's; Alzheimer's risk
In this review focus is on structural imaging in the Alzheimer’s disease pre-states, particularly cognitively normal (CN) persons at future dementia risk. Findings in mild cognitive impairment (MCI) are described here only for comparison with CN. Cited literature evidence and commentary address issues of structural imaging alterations in CN that precede MCI and AD, regional patterns of such alterations, and the time relationship between structural imaging alterations and the appearance of symptoms of AD, issues relevant to the conduct of future AD prevention trials.
review; human; mild cognitive impairment; Alzheimer’s disease; magnetic resonance imaging; morphometry; cognitive normality; normal aging
Molecular chaperones, as the name suggests, are involved in folding, maintenance, intracellular transport, and degradation of proteins as well as in facilitating cell signaling. Heat shock protein 90 (Hsp90) is an essential eukaryotic molecular chaperone that carries out these processes in normal and cancer cells. Hsp90 function in vivo is coupled to its ability to hydrolyze ATP and this can be regulated by co-chaperones and post-translational modifications. In this review, we explore the varied roles of known post-translational modifications of cytosolic and nuclear Hsp90 (phosphorylation, acetylation, S-nitrosylation, oxidation and ubiquitination) in fine-tuning chaperone function in eukaryotes.
Heat Shock Protein 90; Hsp82; Post-translational modification Phosphorylation; Acetylation; Oxidation; Nitrosylation
The apoA-I molecule adopts a two-domain tertiary structure and the properties of these domains modulate the ability to form HDL particles. Thus, human apoA-I differs from mouse apoA-I in that it can form smaller HDL particles; the C-terminal α-helix is important in this process and human apoA-I is unusual in containing aromatic amino acids in the non-polar face of this amphipathic α-helix. To understand the influence of these aromatic amino acids and the associated high hydrophobicity, apoA-I variants were engineered in which aliphatic amino acids were substituted with or without causing a decrease in overall hydrophobicity. The variants human apoA-I (F225L/F229A/Y236A) and apoA-I (F225L/F229L/A232L/Y236L) were compared to wild-type (WT) apoA-I for their abilities to (1) solubilize phospholipid vesicles and form HDL particles of different sizes, and (2) mediate cellular cholesterol efflux and create nascent HDL particles via ABCA1. The loss of aromatic residues and concomitant decrease in hydrophobicity in apoA-I (F225L/F229A/Y236A) has no effect on protein stability, but reduces by a factor of about three the catalytic efficiencies (Vmax/Km) of vesicle solubilization and cholesterol efflux; also, relatively large HDL particles are formed. With apoA-I (F225L/F229L/A232L/Y236L) where the hydrophobicity is restored by the presence of only leucine residues in the helix non-polar face, the catalytic efficiencies of vesicle solubilization and cholesterol efflux are similar to those of WT apoA-I; this variant forms smaller HDL particles. Overall, the results show that the hydrophobicity of the non-polar face of the C-terminal amphipathic α-helix plays a critical role in determining apoA-I functionality but aromatic amino acids are not required.
ATP Binding Cassette Transporter A1 (ABCA1); amphipathic α-helix; apolipoprotein A-I; cellular cholesterol efflux; high density lipoprotein (HDL); lipid solubilization
FACT is a roughly 180 kDa heterodimeric protein complex important for managing the properties of chromatin in eukaryotic cells. Chromatin is a repressive barrier that plays an important role in protecting genomic DNA and regulating access to it. This barrier must be temporarily removed during transcription, replication, and repair, but it also must be rapidly restored to the original state afterwards. Further, the properties of chromatin are dynamic and must be adjusted as conditions dictate. FACT was identified as a factor that destabilizes nucleosomes in vitro, but it has now also been implicated as a central factor in the deposition of histones to form nucleosomes, as an exchange factor that swaps the histones within existing nucleosomes for variant forms, and as a tether that prevents histones from being displaced by the passage of RNA polymerases during transcription. FACT therefore plays central roles in building, maintaining, adjusting, and overcoming the chromatin barrier. This review summarizes recent results that have begun to reveal how FACT can promote what appear to be contradictory goals, using a simple set of binding activities to both enhance and diminish the stability of nucleosomes.
In some settings increasing high density lipoprotein (HDL) levels has been associated with a reduction in experimental atherosclerosis. This has been most clearly seen in apolipoprotein A-I (apoA-I) transgenic mice or in animals infused with HDL or its apolipoproteins. A major mechanism by which these treatments are thought to delay progression or cause regression of atherosclerosis is by promoting efflux of cholesterol from macrophage foam cells. In addition, HDL has been described as having anti-inflammatory and other beneficial effects. Some recent research has linked anti-inflammatory effects to cholesterol efflux pathways but likely multiple mechanisms are involved. Macrophage cholesterol efflux may have a role in facilitating emigration of macrophages from lesions during regression. While macrophages can mediate cholesterol efflux by several pathways, studies in knockout mice or cells point to the importance of active efflux mediated by ATP binding cassette transporter (ABC) A1 and G1. In addition to traditional roles in macrophages, these transporters have been implicated in the control of hematopoietic stem cell proliferation, monocytosis and neutrophilia, as well as activation of monocytes and neutrophils. Thus, HDL and cholesterol efflux pathways may have important anti-atherogenic effects at all stages of the myeloid cell/monocyte/dendritic cell/macrophage lifecycle.
Multicellular organisms regulate the uptake of calories, trace elements, and other nutrients by complex feedback mechanisms. In the case of iron, the body senses internal iron stores, iron requirements for hematopoiesis, and inflammatory status, and regulates iron uptake by modulating the uptake of dietary iron from the intestine. Both the liver and the intestine participate in the coordination of iron uptake and distribution in the body. The liver senses inflammatory signals and iron status of the organism and secretes a peptide hormone, hepcidin. Under high iron or inflammatory conditions hepcidin levels increase. Hepcidin binds to the iron transport protein, ferroportin (FPN), promoting FPN internalization and degradation. Decreased FPN levels reduce iron efflux out of intestinal epithelial cells and macrophages into the circulation. Derangements in iron metabolism result in either the abnormal accumulation of iron in the body, or in anemias. The identification of the mutations that cause the iron overload disease, hereditary hemochromatosis (HH), or iron-refractory iron-deficiencey anemia has revealed many of the proteins used to regulate iron uptake.
Scope of the review
In this review we discuss recent data concerning the regulation of iron homeostasis in the body by the liver and how transferrin receptor 2 (TfR2) affects this process.
TfR2 plays a key role in regulating iron homeostasis in the body.
The regulation of iron homeostasis is important. One third of the people in the world are anemic. HH is the most common inherited disease in people of Northern European origin and can lead to severe health complications if left untreated.
Hereditary hemochromatosis; transferrin receptor 2; TfR2; HFE; hepcidin; hemojuvelin; BMP; ferroportin
White matter (WM) changes measured using diffusion tensor imaging (DTI) have been reported in Alzheimer’s disease (AD) and amnestic mild cognitive impairment (MCI), but changes in earlier pre-MCI stages have not been fully investigated.
In a cross-sectional analysis, older adults with MCI (n=28), older adults with cognitive complaints but without psychometric impairment (CC, n=29) and healthy controls (HC, n=35) were compared. Measures included whole-brain DTI, T1-weighted structural MRI, and neuropsychological assessment. Diffusion images were analyzed using Tract-Based Spatial Statistics (TBSS). Voxel-wise fractional anisotropy (FA) and mean, axial, and radial diffusivity (MD, DA, DR) were assessed and compared between groups. Significant tract clusters were extracted in order to perform further ROI comparisons. Brain volume was estimated using Freesurfer based on T1 structural images.
The MCI group showed lower FA and higher RD than controls in bilateral parahippocampal WM. When comparing extracted diffusivity measurements from bilateral parahippocampal WM clusters, the CC group had values that were intermediate to the MCI and HC groups. Group difference in DTI measures remained significant after controlling for hippocampal atrophy. Across the entire sample, DTI indices in parahippocampal WM were correlated with memory function.
These findings are consistent with previous results showing changes in parahippocampal WM in AD and MCI compared to controls. The intermediate pattern found in the CC group suggests the potential of DTI to contribute to earlier detection of neurodegenerative changes during prodromal stages.
Alzheimer’s disease; Diffusion tensor imaging; Mild cognitive impairment; MRI; Voxel-based method; White matter; Hippocampus; Memory; Fractional anisotropy; Diffusivity
In this article we review recent research on diffusion tensor imaging (DTI) of white matter (WM) integrity and the implications for age-related differences in cognition. Neurobiological mechanisms defined from DTI analyses suggest that a primary dimension of age-related decline in WM is a decline in the structural integrity of myelin, particularly in brain regions that myelinate later developmentally. Research integrating behavioral measures with DTI indicates that WM integrity supports the communication among cortical networks, particularly those involving executive function, perceptual speed, and memory (i.e., fluid cognition). In the absence of significant disease, age shares a substantial portion of the variance associated with the relation between WM integrity and fluid cognition. Current data are consistent with one model in which age-related decline in WM integrity contributes to a decreased efficiency of communication among networks for fluid cognitive abilities. Neurocognitive disorders for which older adults are at risk, such as depression, further modulate the relation between WM and cognition, in ways that are not as yet entirely clear. Developments in DTI technology are providing new insight into both the neurobiological mechanisms of aging WM and the potential contribution of DTI to understanding functional measures of brain activity.
Magnetic resonance imaging; Brain; Behavior; Adult development; Neuroaxonal damage
Quality control processes regulate the proteome by determining whether a protein is to be folded or degraded. Hsp90 is a hub in the network of molecular chaperones that maintain this process because it promotes both folding and degradation, in addition to regulating expression of other quality control components. The significance of Hsp90’s role in quality control is enhanced by the function of its clients, which include protein kinases and transcription factors, in cellular signaling. Inhibition of Hsp90 with small molecules results in rapid degradation of such clients via the ubiquitin/proteasome pathway, and also in induction of the Hsp70 molecular chaperone. These two events result in markedly different outcomes depending on cell type. For tumor cells there is a profound loss of signaling in growth promoting pathways. By contrast, increased amounts of Hsp70 in neuronal cells ameliorate the toxicity that is associated with formation of aggregates observed in neurodegenerative conditions. In this review we discuss the mechanisms underlying these differential effects of Hsp90 inhibition on the quality control of distinct client proteins.
ATP-binding cassette transporter A1 (ABCA1) is an integral cell membrane protein that protects cardiovascular disease by at least two mechanisms: by export of excess cholesterol from cells and by suppression of inflammation. ABCA1 exports cholesterol and phospholipids from cells by multiple steps that involve forming cell surface lipid domains, binding of apolipoproteins to ABCA1, activating signaling pathways, and solubilizing these lipids by apolipoproteins. ABCA1 executes its anti-inflammatory effect by modifying cell membrane lipid rafts and directly activating signaling pathways. The interaction of apolipoproteins with ABCA1 activates multiple signaling pathways, including Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3), protein kinase A, Rho family G protein CDC42 and protein kinase C. Activating protein kinase A and Rho family G protein CDC42 regulates ABCA1-mediated lipid efflux, activating PKC stabilizes ABCA1 protein, and activating JAK2/STAT3 regulates both ABCA1-mediated lipid efflux and anti-inflammation. Thus, ABCA1 behaves both as a lipid exporter and a signaling receptor. Targeting ABCA1 receptor-like property using agonists for ABCA1 protein could become a promising new therapeutic target for increasing ABCA1 function and treating cardiovascular disease.
ATP-binding cassette transporter A1; signaling pathways; cholesterol efflux; lipid metabolism
Aging is associated with appearance of white matter hyperintensities (WMH) on MRI scans. Vascular risk and inflammation, which increase with age, may contribute to white matter deterioration and proliferation of WMH. We investigated whether circulating biomarkers and genetic variants associated with elevated vascular risk and inflammation are associated with WMH volume in healthy adults (144 volunteers, 44-77 years of age). We examined association of WMH volume with age, sex, hypertension, circulating levels of total plasma homocysteine (tHcy), cholesterol (low-density lipoprotein), and C-reactive protein (CRP), and four polymorphisms related to vascular risk and inflammation: Apolipoprotein ε (ApoE ε2,3,4), Angiotensin-Converting Enzyme insertion/deletion (ACE I/D), methylenetetrahydrofolate reductase (MTHFR) C677T, C-reactive protein (CRP) -286 C>A>T, and interleukin-1β (IL-1β) C-511T. We found that larger WMH volume was associated with advanced age, hypertension, and elevated levels of homocysteine and CRP but not with low-density lipoprotein levels. Homozygotes for IL-1β -511T allele and carriers of CRP -286T allele that are associated with increased inflammatory response had larger WMH than the other allelic combinations. Carriers of the APOE ε2 allele had larger frontal WMH than ε3 homozygotes and ε4 carriers did. Thus, in healthy adults, who are free of neurological and vascular disease, genetic variants that promote inflammation and elevated levels of vascular risk biomarkers can contribute to brain abnormalities.
brain; aging; CRP; interleukin; homocysteine; frontal lobe; ApoE; MTHFR; ACE
Human serum transferrin (hTF) is a bilobal glycoprotein that reversibly binds Fe3+ and delivers it to cells by the process of receptor-mediated endocytosis. Despite decades of research, the precise events resulting in iron release from each lobe of hTF within the endosome have not been fully delineated.
Scope of Review
We provide an overview of the kinetics of iron release from hTF ± the transferrin receptor (TFR) at endosomal pH (5.6). A critical evaluation of the array of biophysical techniques used to determine accurate rate constants is provided.
Delivery of Fe3+ by to actively dividing cells by hTF is essential; too much or too little Fe3+ directly impacts the well-being of an individual. Because the interaction of hTF with the TFR controls iron distribution in the body, an understanding of this process at the molecular level is essential.
Not only does TFR direct the delivery of iron to the cell through the binding of hTF, kinetic data demonstrate that it also modulates iron release from the N- and C-lobes of hTF. Specifically, the TFR balances the rate of iron release from each lobe, resulting in efficient Fe3+ release within a physiologically relevant time frame.
transferrin; transferrin receptor; kinetics; fluorescence
Not long after the Big Bang, iron began to play a central role in the Universe and soon became mired in the tangle of biochemistry that is the prima essentia of life. Since life’s addiction to iron transcends the oxygenation of the Earth’s atmosphere, living things must be protected from the potentially dangerous mix of iron and oxygen. The human being possesses grams of this potentially toxic transition metal, which is shuttling through his oxygen-rich humor. Since long before the birth of modern medicine, the blood—vibrant red from a massive abundance of hemoglobin iron—has been a focus for health experts.
Scope of Review
We describe the current understanding of iron metabolism, highlight the many important discoveries that accreted this knowledge, and describe the perils of dysfunctional iron handling.
Isaac Newton famously penned, “If I have seen further than others, it is by standing upon the shoulders of giants”. We hope that this review will inspire future scientists to develop intellectual pursuits by understanding the research and ideas from many remarkable thinkers of the past.
The history of iron research is a long, rich story with early beginnings, and is far from being finished.
Protein folding quality control does not occur randomly in cells, but requires the action of specialized molecular chaperones compartmentalized in subcellular microenvironments and organelles. Fresh experimental evidence has now linked a mitochondrial-specific Heat Shock Protein-90 (Hsp90) homolog, Tumor Necrosis Factor Receptor-Associated Protein-1 (TRAP-1) to pleiotropic signaling circuitries of organelle integrity and cellular homeostasis. TRAP-1-directed compartmentalized protein folding is broadly exploited in cancer and neurodegenerative diseases, presenting new opportunities for therapeutic intervention in humans.
Mitochondria; apoptosis; permeability transition pore; chaperone; tumor growth
We assessed the relationship of insulin resistance with cognitive decline and brain atrophy over two years in early Alzheimer’s disease (AD, n=48) and nondemented controls (n=61). Intravenous glucose tolerance tests were conducted at baseline to determine insulin area-under-the-curve (AUC). A standard battery of cognitive tasks and MRI were conducted at baseline and 2-year follow-up. In nondemented controls, higher baseline insulin AUC was associated with 2-year decline in global cognitive performance (beta=−0.36, p=0.005). In early AD, however, higher insulin AUC was associated with less decline in global cognitive performance (beta=0.26, p=0.06), slower global brain atrophy (beta=0.40, p=0.01) and less regional atrophy in the bilateral hippocampi and cingulate cortices. While insulin resistance is associated with cognitive decline in nondemented aging, higher peripheral insulin may have AD-specific benefits or insulin signaling may be affected by systemic physiologic changes associated with AD.
The concept of reserve arose from the mismatch between the extent of brain changes or pathology and the clinical manifestations of these brain changes. The cognitive reserve hypothesis posits that individual differences in the flexibility and adaptability of brain networks underlying cognitive function may allow some people to cope better with brain changes than others. Although there is ample epidemiologic evidence for cognitive reserve, the neural substrate of reserve is still a topic of ongoing research. Here we review some representative studies from our group that exemplify possibilities for the neural substrate of reserve including neural reserve, neural compensation, and generalized cognitive reserve networks. We also present a schematic overview of our ongoing research in this area.
We report a systems genetics analysis of high density lipoproteins (HDL) levels in an F2 intercross between inbred strains CAST/EiJ and C57BL/6J. We previously showed that there are dramatic differences in HDL metabolism in a cross between these strains, and we now report co-expression network analysis of HDL that integrates global expression data from liver and adipose with relevant metabolic traits. Using data from a total of 293 F2 intercross mice, we constructed weighted gene co-expression networks and identified modules (subnetworks) associated with HDL and clinical traits. These were examined for genes implicated in HDL levels based on large human genome-wide associations studies (GWAS) and examined with respect to conservation between tissue and sexes in a total of 9 data sets. We identify genes that are consistently ranked high by association with HDL across the 9 data sets. We focus in particular on two genes, Wfdc2 and Hdac3, that are located in close proximity to HDL QTL peaks where causal testing indicates that they may affect HDL. Our results provide a rich resource for studies of complex metabolic interactions involving HDL.
Diabetes and insulin resistance increase the risk of cardiovascular disease caused by atherosclerosis through mechanisms that are poorly understood. Lipid-loaded macrophages are key contributors to all stages of atherosclerosis. We have recently shown that diabetes associated with increased plasma lipids reduces cholesterol efflux and levels of the reverse cholesterol exporter ABCA1 (ATP-binding cassette transporter A1) in mouse macrophages, which likely contributes to macrophage lipid accumulation in diabetes. Furthermore, we and others have shown that unsaturated fatty acids reduce ABCA1-mediated cholesterol efflux, and that this effect is mediated by the acyl-CoA derivatives of the fatty acids. We therefore investigated whether acyl-CoA synthetase 1 (ACSL1), a key enzyme mediating acyl-CoA synthesis in macrophages, could directly influence ABCA1 levels and cholesterol efflux in these cells. Mouse macrophages deficient in ACSL1 exhibited reduced sensitivity to oleate- and linoleate-mediated ABCA1 degradation, which resulted in increased ABCA1 levels and increased apolipoprotein A-I-dependent cholesterol efflux in the presence of these fatty acids, as compared with wildtype mouse macrophages. Conversely, overexpression of ACSL1 resulted in reduced ABCA1 levels and reduced cholesterol efflux in the presence of unsaturated fatty acids. Thus, the reduced ABCA1 and cholesterol efflux in macrophages subjected to conditions of diabetes and elevated fatty load may, at least in part, be mediated by ACSL1. These observations raise the possibility that ABCA1 levels could be increased by inhibition of acyl-CoA synthetase activity in vivo.
acyl-CoA synthetase; ATP-binding cassette transporter A1; cholesterol efflux; high-density lipoprotein; lipid metabolism; macrophage
Extracellular Hsp90 proteins, including “membrane-bound”, “released” and “secreted”, were first reported more than two decades ago. Only studies of the past seven years have begun to reveal a picture for when, how and why Hsp90 get exported by both normal and tumor cells. Normal cells secrete Hsp90 in response to tissue injury. Tumor cells have managed to constitutively secrete Hsp90 for tissue invasion. In either case, sufficient supply of the extracellular Hsp90 can be guaranteed by its unusually abundant storage inside the cells. A well-characterized function of secreted Hsp90α is to promote cell motility, a crucial event for both wound healing and cancer. The reported targets for extracellular Hsp90α include MMP2, LRP-1, tyrosine kinase receptors and possibly more. The pro-motility activity of secreted Hsp90α resides within a fragment at the boundary between linker region and middle domain. Inhibition of its secretion, neutralization of its extracellular action or interruption of its signaling through LRP-1 block wound healing and tumor invasion in vitro and in vivo. In normal tissue, topical application of F-5 promotes acute and diabetic wound healing far more effectively than US FDA-approved conventional growth factor therapy in mice. In cancer, drugs that selectively target the F-5 region of secreted Hsp90 by cancer cells may be more effective and less toxic than those that target the ATPase of the intracellular Hsp90.