Genetic inactivation of the allograft inflammatory factor‐1 locus

Allograft inflammatory factor‐1 (Aif‐1) is a 17 kDa EF hand motif‐bearing protein expressed primarily in developing spermatids and cells of monocyte/macrophage lineage. Increased Aif‐1 expression has been identified in clinically important conditions, including rheumatoid arthritis, systemic sclerosis, endometriosis, and transplant‐associated arteriosclerosis. Largely similar gene products arising from the same locus are known as ionized Ca²⁺ binding adapter‐1 (Iba1), microglial response factor‐1 (MRF1), and daintain; Iba1 in particular has emerged as a histologic marker of microglia and their activation in pathologic CNS conditions, including the response to facial nerve axotomy and stroke, uveitis, and experimental autoimmune neuritis and encephalomyelitis. Nevertheless, how aif‐1 gene products affect cellular function is only partly understood, and the physiologic significance of these products for male fertility, immune system development, and inflammation has not been described. To permit such investigations, we generated a mouse line with targeted deletion of the coding regions of the aif‐1 gene. Here we report that mice lacking Aif‐1 breed well and show normal post‐natal growth, but show resistance to disease in a model of collagen‐induced arthritis. We anticipate that these mice will be useful for studies of Aif‐1 function in a variety of immune and inflammatory disease models. genesis 51:734–740. © 2013 Wiley Periodicals, Inc.     

Administration of DHA Reduces Endoplasmic Reticulum Stress-Associated Inflammation and Alters Microglial or Macrophage Activation in Traumatic Brain Injury

We investigated the effects of the administration of docosahexaenoic acid (DHA) post-traumatic brain injury (TBI) on reducing neuroinflammation. TBI was induced by cortical contusion injury in Sprague Dawley rats. Either DHA (16 mg/kg in dimethyl sulfoxide) or vehicle dimethyl sulfoxide (1 ml/kg) was administered intraperitonially at 5 min after TBI, followed by a daily dose for 3 to 21 days. TBI triggered activation of microglia or macrophages, detected by an increase of Iba1 positively stained microglia or macrophages in peri-lesion cortical tissues at 3, 7, and 21 days post-TBI. The inflammatory response was further characterized by expression of the proinflammatory marker CD16/32 and the anti-inflammatory marker CD206 in Iba1⁺ microglia or macrophages. DHA-treated brains showed significantly fewer CD16/32⁺ microglia or macrophages, but an increased CD206⁺ phagocytic microglial or macrophage population. Additionally, DHA treatment revealed a shift in microglial or macrophage morphology from the activated, amoeboid-like state into the more permissive, surveillant state. Furthermore, activated Iba1⁺ microglial or macrophages were associated with neurons expressing the endoplasmic reticulum (ER) stress marker CHOP at 3 days post-TBI, and the administration of DHA post-TBI concurrently reduced ER stress and the associated activation of Iba1⁺ microglial or macrophages. There was a decrease in nuclear translocation of activated nuclear factor kappa-light-chain-enhancer of activated B cells protein at 3 days in DHA-treated tissue and reduced neuronal degeneration in DHA-treated brains at 3, 7, and 21 days after TBI. In summary, our study demonstrated that TBI mediated inflammatory responses are associated with increased neuronal ER stress and subsequent activation of microglia or macrophages. DHA administration reduced neuronal ER stress and subsequent association with microglial or macrophage polarization after TBI, demonstrating its therapeutic potential to ameliorate TBI-induced cellular pathology.     

miR‐124 Contributes to the functional maturity of microglia

During early development of the central nervous system (CNS), a subset of yolk‐sac derived myeloid cells populate the brain and provide the seed for the microglial cell population, which will self‐renew throughout life. As development progresses, individual microglial cells transition from a phagocytic amoeboid state through a transitional morphing phase into the sessile, ramified, and normally nonphagocytic microglia observed in the adult CNS under healthy conditions. The molecular drivers of this tissue‐specific maturation profile are not known. However, a survey of tissue resident macrophages identified miR‐124 to be expressed in microglia. In this study, we used transgenic zebrafish to overexpress miR‐124 in the mpeg1 expressing yolk‐sac‐derived myeloid cells that seed the microglia. In addition, a systemic sponge designed to neutralize the effects of miR‐124 was used to assess microglial development in a miR‐124 loss‐of‐function environment. Following the induction of miR‐124 overexpression, microglial motility and phagocytosis of apoptotic cells were significantly reduced. miR‐124 overexpression in microglia resulted in the accumulation of residual apoptotic cell bodies in the optic tectum, which could not be achieved by miR‐124 overexpression in differentiated neurons. Conversely, expression of the miR‐124 sponge caused an increase in the motility of microglia and transiently rescued motility and phagocytosis functions when activated simultaneously with miR‐124 overexpression. This study provides in vivo evidence that miR‐124 activity has a key role in the development of functionally mature microglia. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 507–518, 2016     

Allograft inflammatory factor-1/Ionized calcium-binding adapter molecule 1 is specifically expressed by most subpopulations of macrophages and spermatids in testis

Ionized calcium-binding adapter molecule 1 (Iba1) is a 147-amino-acid calcium-binding protein widely in use as a marker for microglia. It has actin-crosslinking activity and is involved in aspects of motility-associated rearrangement of the actin cytoskeleton. The Iba1 gene and protein are identical to allograft inflammatory factor-1 (AIF-1), a protein involved in various aspects of inflammation, which was investigated independently from Iba1. Although regarded to be monocyte/macrophage-specific, expression by germ cells in testis showed that AIF-1/Iba1 is not exclusively expressed by cells of the monocyte/macrophage lineage. Furthermore, AIF-1 was found in cells not belonging to the monocyte/macrophage lineage under pathological conditions. Here, the distribution of AIF-1/Iba1 in the normal mouse has been examined, by immunohistochemistry, to determine whether AIF-1/Iba1 expression is confined to macrophages and spermatids. Spermatids are the only cells not belonging to the monocyte/macrophage lineage found to express AIF-1/Iba1 in the normal mouse, by this method. This study has not demonstrated AIF-1/Iba1 expression in dendritic cells, although this protein might be expressed by subsets of dendritic cells. AIF-1/Iba1 can be regarded a “pan-macrophage marker” because, except for alveolar macrophages, all subpopulations of macrophages examined express AIF-1/Iba1.     

Molecular mechanisms of apoptosis induced by ajoene in 3T3-L1 adipocytes

Objective : Determine the biochemical pathways involved in induction of apoptosis by ajoene, an organosulfur compound from garlic. Research Methods and Procedures : Mature 3T3‐L1 adipocytes were incubated with ajoene at concentrations up to 200 μM. Viability and apoptosis were quantified using an MTS‐based cell viability assay and an enzyme‐linked immunosorbent assay for single‐stranded DNA (ssDNA), respectively. Intracellular reactive oxygen species (ROS) production was measured based on production of the fluorescent dye, dichlorofluorescein. Activation of the mitogen‐activated protein kinases extracellular signal‐regulating kinase 1/2 (ERK) and c‐Jun‐N‐terminal kinase (JNK) was shown by Western blot. Western blot was also used to show activation of caspase‐3, translocation of apoptosis‐inducing factor (AIF) from mitochondria to nucleus, and cleavage of 116‐kDa poly(ADP‐ribose) polymerase (PARP)‐1. Results : Ajoene induced apoptosis of 3T3‐L1 adipocytes in a dose‐ and time‐dependent manner. Ajoene treatment resulted in activation of JNK and ERK, translocation of AIF from mitochondria to nucleus, and cleavage of 116‐kDa PARP‐1 in a caspase‐independent manner. Ajoene treatment also induced an increase in intracellular ROS level. Furthermore, the antioxidant N‐acetyl‐l ‐cysteine effectively blocked ajoene‐mediated ROS generation, activation of JNK and ERK, translocation of AIF, and degradation of PARP‐1. Discussion : These results indicate that ajoene‐induced apoptosis in 3T3‐L1 adipocytes is initiated by the generation of hydrogen peroxide, which leads to activation of mitogen‐activated protein kinases, degradation of PARP‐1, translocation of AIF, and fragmentation of DNA. Ajoene can, thus, influence the regulation of fat cell number through the induction of apoptosis and may be a new therapeutic agent for the treatment of obesity.     

Prevention of inflammation‐mediated neurotoxicity by butylidenephthalide and its role in microglial activation

Microglial cells are the prime effectors in immune and inflammatory responses of the central nervous system (CNS). During pathological conditions, the activation of these cells helps restore CNS homeostasis. However, chronic microglial activation endangers neuronal survival through the release of various proinflammatory molecules and neurotoxins. Thus, negative regulators of microglial activation have been considered as potential therapeutic candidates to target neurodegeneration, such as that in Alzheimer's and Parkinson's diseases. The rhizome of Ligusticum chuanxiong Hort. (Ligusticum wallichii Franch) has been widely used for the treatment of vascular diseases in traditional oriental medicine. Butylidenephthalide (BP), a major bioactive component from L. chuanxiong, has been reported to have a variety of pharmacological activities, including vasorelaxant, anti‐anginal, anti‐platelet and anti‐cancer effects. The aim of this study was to examine whether BP represses microglial activation. In rat brain microglia, BP significantly inhibited the lipopolysaccharide (LPS)‐induced production of nitric oxide (NO), tumour necrosis factor‐α and interleukin‐1β. In organotypic hippocampal slice cultures, BP clearly blocked the effect of LPS on hippocampal cell death and inhibited LPS‐induced NO production in culture medium. These results newly suggest that BP provide neuroprotection by reducing the release of various proinflammatory molecules from activated microglia. Copyright © 2013 John Wiley & Sons, Ltd.     

A synthetic peptide corresponding to a region of the human pericentriolar material 1 (PCM‐1) protein binds β‐amyloid (Aβ1‐42) oligomers

We have recently reported that a ~19‐kDa polypeptide, rPK‐4, is a protein kinase Cs inhibitor that is 89% homologous to the 1171–1323 amino acid region of the 228‐kDa human pericentriolar material‐1 (PCM‐1) protein (Chakravarthy et al. 2012). We have now discovered that rPK‐4 binds oligomeric amyloid‐β peptide (Aβ)_1‐42 with high affinity. Most importantly, a PCM‐1‐selective antibody co‐precipitated Aβ and amyloid β precursor protein (AβPP) from cerebral cortices and hippocampi from AD (Alzheimer's disease) transgenic mice that produce human AβPP and Aβ_1‐42, suggesting that PCM‐1 may interact with amyloid precursor protein/Aβ in vivo. We have identified rPK‐4′s Aβ‐binding domain using a set of overlapping synthetic peptides. We have found with ELISA, dot‐blot, and polyacrylamide gel electrophoresis techniques that a ~ 5 kDa synthetic peptide, amyloid binding peptide (ABP)‐p4‐5 binds Aβ_1‐42 at nM levels. Most importantly, ABP‐p4‐5, like rPK‐4, appears to preferentially bind Aβ_1‐42 oligomers, believed to be the toxic AD‐drivers. As expected from these observations, ABP‐p4‐5 prevented Aβ_1‐42 from killing human SH‐SY5Y neuroblastoma cells via apoptosis. These findings indicate that ABP‐p4‐5 is a possible candidate therapeutic for AD.     

5‐Aminoimidazole‐4‐carboxamide ribonucleoside‐mediated adenosine monophosphate‐activated protein kinase activation induces protective innate responses in bacterial endophthalmitis

The retina is considered to be the most metabolically active tissue in the body. However, the link between energy metabolism and retinal inflammation, as incited by microbial infection such as endophthalmitis, remains unexplored. In this study, using a mouse model of Staphylococcus aureus (SA) endophthalmitis, we demonstrate that the activity (phosphorylation) of 5' adenosine monophosphate‐activated protein kinase alpha (AMPKα), a cellular energy sensor and its endogenous substrate; acetyl‐CoA carboxylase is down‐regulated in the SA‐infected retina. Intravitreal administration of an AMPK activator, 5‐aminoimidazole‐4‐carboxamide ribonucleoside (AICAR), restored AMPKα and acetyl‐CoA carboxylase phosphorylation. AICAR treatment reduced both the bacterial burden and intraocular inflammation in SA‐infected eyes by inhibiting NF‐kB and MAP kinases (p38 and JNK) signalling. The anti‐inflammatory effects of AICAR were diminished in eyes pretreated with AMPK inhibitor, Compound C. The bioenergetics (Seahorse) analysis of SA‐infected microglia and bone marrow‐derived macrophages revealed an increase in glycolysis, which was reinstated by AICAR treatment. AICAR also reduced the expression of SA‐induced glycolytic genes, including hexokinase 2 and glucose transporter 1 in microglia, bone marrow‐derived macrophages and the mouse retina. Interestingly, AICAR treatment enhanced the bacterial phagocytic and intracellular killing activities of cultured microglia, macrophages and neutrophils. Furthermore, AMPKα1 global knockout mice exhibited increased susceptibility towards SA endophthalmitis, as evidenced by increased inflammatory mediators and bacterial burden and reduced retinal function. Together, these findings provide the first evidence that AMPK activation promotes retinal innate defence in endophthalmitis by modulating energy metabolism and that it can be targeted therapeutically to treat ocular infections.     

Molecular cloning and characterization of LKv1, a novel voltage‐gated potassium channel in leech

We have cloned a novel voltage‐gated K channel, LKv1, in two species of leech. The properties of LKv1 expressed in transiently transfected HEK293 cells is that of a delayed rectifier current. LKv1 may be a major modulator of excitability in leech neurons, since antibody localization studies show that LKv1 is expressed in the soma and axons of all neurons in both the central and peripheral nervous systems. Comparison of the biophysical and pharmacological properties of LKv1 with native voltage‐gated conductances in leech neurons suggests that LKv1 may correspond to the previously characterized delayed rectifier current, I_K. Phylogenetic analysis of LKv1 shows that it is related to the Shaker subfamily of voltage‐gated K channels although it occupies a separate branch from that of the monophyletic Shaker clade composed of the flatworm, Aplysia, Drosophila, and mammalian Shaker homologs as well as from that of two recently identified Shaker‐related K channels in jellyfish. Thus, this analysis indicates that this group of voltage‐gated K channels contains several evolutionarily divergent lineages. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 287–299, 1999     

X-ray structures of the microglia/macrophage-specific protein Iba1 from human and mouse demonstrate novel molecular conformation change induced by calcium binding

The ionized calcium-binding adaptor molecule 1 (Iba1) with 147 amino acid residues has been identified as a calcium-binding protein, expressed specifically in microglia/macrophages, and is expected to be a key factor in membrane ruffling, which is a typical feature of activated microglia. We have determined the crystal structure of human Iba1 in a Ca(2+)-free form and mouse Iba1 in a Ca(2+)-bound form, to a resolution of 1.9 A and 2.1 A, respectively. X-ray structures of Iba1 revealed a compact, single-domain protein with two EF-hand motifs, showing similarity in overall topology to partial structures of the classical EF-hand proteins troponin C and calmodulin. In mouse Iba1, the second EF-hand contains a bound Ca(2+), but the first EF-hand does not, which is often the case in S100 proteins, suggesting that Iba1 has S100 protein-like EF-hands. The molecular conformational change induced by Ca(2+)-binding of Iba1 is different from that found in the classical EF-hand proteins and/or S100 proteins, which demonstrates that Iba1 has an unique molecular switching mechanism dependent on Ca(2+)-binding, to interact with target molecules.     

Diagnostic value of serum concentrations of high‐mobility group‐box protein 1 and soluble hemoglobin scavenger receptor in brucellosis

Both cluster of differentiation (CD)4⁺ and CD8⁺ T lymphocytes play key roles in immunity to Brucella, in part because they secrete interferon (IFN)‐γ and activate bactericidal functions in macrophages. Therefore, use of markers of macrophage activation may have diagnostic and prognostic significance. High‐mobility group‐box 1 protein (HMGB1), a late‐onset pro‐inflammatory cytokine, is secreted by activated macrophages. Soluble hemoglobin scavenger receptor (sCD163) is a specific marker of anti‐inflammatory macrophages. The aim of this study was to investigate the diagnostic value of HMGB1 and sCD163 concentrations in brucellosis and its various clinical forms. Serum HMGB1 and sCD163 concentrations in 49 brucellosis patients were compared with those in 52 healthy control subjects. Both serum HMGB1 and sCD163 concentrations were significantly higher in brucellosis patients than in healthy controls (P < 0.001). There were no statistically significant differences in serum concentrations of HMGB1 and sCD163 between cases of acute, subacute and chronic brucellosis. Additionally, serum HMGB1 concentrations were positively correlated with sCD163 concentrations, whereas neither HMGB1 nor sCD163 concentrations were correlated with C‐reactive protein concentrations, white cell counts or erythrocyte sedimentation rates. Therefore, serum concentrations of HMGB1 and sCD163 may be diagnostic markers for brucellosis, but neither can be used to differentiate the three different forms of this disease (acute, subacute and chronic).     

Reactive oxygen species‐induced cell death of rat primary astrocytes through mitochondria‐mediated mechanism

Astrocytes, the most abundant glial cell population in the central nervous system (CNS), play physiological roles in neuronal activities. Oxidative insult induced by the injury to the CNS causes neural cell death through extrinsic and intrinsic pathways. This study reports that reactive oxygen species (ROS) generated by exposure to the strong oxidizing agent, hexavalent chromium (Cr(VI)) as a chemical‐induced oxidative stress model, caused astrocytes to undergo an apoptosis‐like cell death through a caspase‐3‐independent mechanism. Although activating protein‐1 (AP‐1) and NF‐κB were activated in Cr(VI)‐primed astrocytes, the inhibition of their activity failed to increase astrocytic cell survival. The results further indicated that the reduction in mitochondrial membrane potential (MMP) was accompanied by an increase in the levels of ROS in Cr(VI)‐primed astrocytes. Moreover, pretreatment of astrocytes with N‐acetylcysteine (NAC), the potent ROS scavenger, attenuated ROS production and MMP loss in Cr(VI)‐primed astrocytes, and significantly increased the survival of astrocytes, implying that the elevated ROS disrupted the mitochondrial function to result in the reduction of astrocytic cell viability. In addition, the nuclear expression of apoptosis‐inducing factor (AIF) and endonuclease G (EndoG) was observed in Cr(VI)‐primed astrocytes. Taken together, evidence shows that astrocytic cell death occurs by ROS‐induced oxidative insult through a caspase‐3‐independent apoptotic mechanism involving the loss of MMP and an increase in the nuclear levels of mitochondrial pro‐apoptosis proteins (AIF/EndoG). This mitochondria‐mediated but caspase‐3‐independent apoptotic pathway may be involved in oxidative stress‐induced astrocytic cell death in the injured CNS. J. Cell. Biochem. 107: 933–943, 2009. © 2009 Wiley‐Liss, Inc.     

Proteomic characterization of human proinflammatory M1 and anti‐inflammatory M2 macrophages and their response to Candida albicans

In response to different stimuli, macrophages can differentiate into either a pro‐inflammatory subtype (M1, classically activated macrophages) or acquire an anti‐inflammatory phenotype (M2, alternatively activated macrophages). Candida albicans is the most important opportunistic fungus in nosocomial infections, and it is contended by neutrophils and macrophages during the first steps of the invasive infection. Murine macrophages responses to C. albicans have been widely studied, whereas the responses of human‐polarized macrophages remain less characterized. In this study, we have characterized the proteomic differences between human M1‐ and M2‐polarized macrophages, both in basal conditions and in response to C. albicans, by quantitative proteomics (2DE). This proteomic approach allowed us to identify metabolic routes and cytoskeletal rearrangement components that are the most relevant differences between M1 and M2 macrophages. The analysis has revealed fructose‐1,6‐bisphosphatase 1, a critical enzyme in gluconeogenesis, up‐regulated in M1, as a novel protein marker for macrophage polarization. Regarding the response to C. albicans, an M1‐to‐M2 switch in polarization was observed. This M1‐to‐M2 switch might contribute to Candida pathogenicity by decreasing the generation of specific immune responses, thus enhancing fungal survival and colonization, or instead, may be part of the host attempt to reduce the inflammation and limit the damage of the infection.     

Protein 4.1 G localizes in rodent microglia

Although it was reported that protein 4.1 G, a cytoskeletal protein characterized by its general expression in the body, interacts with some signal transduction molecules in the central nervous system (CNS), its distribution and significance in vivo remained to be elucidated. In the present study, we have identified 4.1 G-positive cells in the rodent CNS, and demonstrated its immunolocalization in the developing mouse CNS. In the rodent CNS, 4.1 G was colocalized with markers for microglia, such as CD45, OX-42 and ionized calcium-binding adapter molecule 1 (Iba1), but not with markers for neuronal or other glial cells. Additionally, colocalization of 4.1 G and A1 adenosine receptor was observed in the mouse cerebrum. In a mixed glial culture, most OX-42-positive microglia were positive for 4.1 G, and 4.1 G isoforms of the same molecular weight as in the rat brain were expressed in cultured microglia, where 4.1 G mRNA was detected by RT-PCR. In the developing mouse cerebral cortex, 4.1 G was detected in immature microglia, which were positive for Iba1. These results indicate that 4.1 G in the CNS is mainly distributed in microglia in vivo. Considering the interactions between 4.1 G and the signal transduction molecules, putative roles have been propsed for 4.1 G in microglial functions in the CNS.     

Reactive oxygen species‐mediated endoplasmic reticulum stress response induces apoptosis of Mycobacterium avium‐infected macrophages by activating regulated IRE1‐dependent decay pathway

Mycobacterium avium, a slow‐growing nontuberculous mycobacterium, causes fever, diarrhoea, loss of appetite, and weight loss in immunocompromised people. We have proposed that endoplasmic reticulum (ER) stress‐mediated apoptosis plays a critical role in removing intracellular mycobacteria. In the present study, we investigated the role of the regulated IRE1‐dependent decay (RIDD) pathway in macrophages during M. avium infection based on its role in the regulation of gene expression. The inositol‐requiring enzyme 1 (IRE1)/apoptosis signal‐regulating kinase 1 (ASK1)/c‐Jun N‐terminal kinase (JNK) signalling pathway was activated in macrophages after infection with M. avium. The expression of RIDD‐associated genes, such as Bloc1s1 and St3gal5, was decreased in M. avium‐infected macrophages. Interestingly, M. avium‐induced apoptosis was significantly suppressed by pretreatment with irestatin (inhibitor of IRE1α) and 4μ8c (RIDD blocker). Macrophages pretreated with N‐acetyl cysteine (NAC) showed decreased levels of reactive oxygen species (ROS), IRE1α, and apoptosis after M. avium infection. The expression of Bloc1s1 and St3gal5 was increased in NAC‐pretreated macrophages following infection with M. avium. Growth of M. avium was significantly increased in irestatin‐, 4μ8c‐, and NAC‐treated macrophages compared with the control. The data indicate that the ROS‐mediated ER stress response induces apoptosis of M. avium‐infected macrophages by activating IRE1α‐RIDD. Thus, activation of IRE1α suppresses the intracellular survival of M. avium in macrophages.     

Insulin induces the low density lipoprotein receptor‐related protein 1 (LRP1) degradation by the proteasomal system in J774 macrophage‐derived cells

Low‐density lipoprotein receptor‐related protein 1 (LRP1) is an endocytic receptor, which binds and internalizes diverse ligands such as activated α₂‐macroglobulin (α₂M*). LRP1 promotes intracellular signaling, which downstream mediates cellular proliferation and migration of different types of cells, including macrophages. Unlike the LDL receptor, LRP1 expression is not sensitive to cellular cholesterol levels but appears to be responsive to insulin. It has been previously demonstrated that insulin increases the cell surface presentation of LRP1 in adipocytes and hepatocytes, which is mediated by the intracellular PI₃K/Akt signaling activation. The LRP1 protein distribution is similar to other insulin‐regulated cell surface proteins, including transferring receptor (Tfr). However, in macrophages, the insulin effect on the LRP1 distribution and expression is not well characterized. Considering that macrophages play a central role in the pathogenesis of atherosclerosis, herein we evaluate the effect of insulin on the cellular expression of LRP1 in J774 macrophages‐derived cells using Western blot and immunofluorescence microscopy. Our data demonstrate that insulin induces a significant decrease in the LRP1 protein content, without changing the specific mRNA level of this receptor. Moreover, insulin specifically affected the protein expression of LRP1 but not Tfr. The insulin‐induced protein degradation of LRP1 in J774 cells was mediated by the activation of the PI₃K/Akt pathway and proteasomal system by an enhanced ubiquitin–receptor conjugation. The decreased content of LRP1 induced by insulin affected the cellular internalization of α₂M*. Thus, we propose that the protein degradation of LRP‐1 induced by insulin in macrophages could have important effects on the pathogenesis of atherosclerosis. J. Cell. Biochem. 106: 372–380, 2009. © 2008 Wiley‐Liss, Inc.