Systemic adiponectin malfunction as a risk factor for cardiovascular disease.

Adiponectin (Ad) is an abundant protein hormone regulatory of numerous metabolic processes. The 30 kDa protein originates from adipose tissue, with full-length and globular domain circulatory forms. A collagenous domain within Ad leads to spontaneous self-assemblage into various oligomeric isoforms, including trimers, hexamers, and high-molecular-weight multimers. Two membrane-spanning receptors for Ad have been identified, with differing concentration distribution in various body tissues. The major intracellular pathway activated by Ad includes phosphorylation of AMP-activated protein kinase, which is responsible for many of Ad\u27s metabolic regulatory, anti-inflammatory, vascular protective, and anti-ischemic properties. Additionally, several AMP-activated protein kinase-independent mechanisms responsible for Ad\u27s anti-inflammatory and anti-ischemic (resulting in cardioprotective) effects have also been discovered. Since its 1995 discovery, Ad has garnered considerable attention for its role in diabetic and cardiovascular pathology. Clinical observations have demonstrated the association of hypoadiponectinemia in patients with obesity, cardiovascular disease, and insulin resistance. In this review, we elaborate currently known information about Ad malfunction and deficiency pertaining to cardiovascular disease risk (including atherosclerosis, endothelial dysfunction, and cardiac injury), as well as review evidence supporting Ad resistance as a novel risk factor for cardiovascular injury, providing insight about the future of Ad research and the protein\u27s potential therapeutic benefits

Effects of subducted seamounts on megathrust earthquake nucleation and rupture propagation

Author Posting. © American Geophysical Union, 2012. This article is posted here by permission of American Geophysical Union for personal use, not for redistribution. The definitive version was published in Geophysical Research Letters 39 (2012): L24302, doi:10.1029/2012GL053892.Subducted seamounts have been linked to interplate earthquakes, but their specific effects on earthquake mechanism remain controversial. A key question is under what conditions a subducted seamount will generate or stop megathrust earthquakes. Here we show results from numerical experiments in the framework of rate- and state-dependent friction law in which a seamount is characterized as a patch of elevated effective normal stress on the thrust interface. We find that whether subducted seamounts generate or impede megathrust earthquakes depends critically on their relative locations to the earthquake nucleation zone defined by depth-variable friction parameters. A seamount may act as a rupture barrier and such barrier effect is most prominent when the seamount sits at an intermediate range of the seamount-to-trench distances (20–100% of the nucleation-zone-to-trench distance). Moreover, we observe that seamount-induced barriers can turn into asperities on which megathrust earthquakes can nucleate at shallow depths and rupture the entire seismogenic zone. These results suggest that a strong barrier patch may not necessarily reduce the maximum size of earthquakes. Instead, the barrier could experience large coseismic slip when it is ruptured.This work is supported by the NSF Grant EAR-1015221 and WHOI Deep Ocean Exploration Institute awards 27071150 and 25051162.2013-06-1

Mechanistic insights revealed by lipid profiling in monogenic insulin resistance syndromes.

BACKGROUND: Evidence from several recent metabolomic studies suggests that increased concentrations of triacylglycerols with shorter (14-16 carbon atoms), saturated fatty acids are associated with insulin resistance and the risk of type 2 diabetes. Although causality cannot be inferred from association studies, patients in whom the primary cause of insulin resistance can be genetically defined offer unique opportunities to address this challenge. METHODS: We compared metabolite profiles in patients with congenital lipodystrophy or loss-of-function insulin resistance (INSR gene) mutations with healthy controls. RESULTS: The absence of significant differences in triacylglycerol species in the INSR group suggest that changes previously observed in epidemiological studies are not purely a consequence of insulin resistance. The presence of triacylglycerols with lower carbon numbers and high saturation in patients with lipodystrophy suggests that these metabolite changes may be associated with primary adipose tissue dysfunction. The observed pattern of triacylglycerol species is indicative of increased de novo lipogenesis in the liver. To test this we investigated the distribution of these triacylglycerols in lipoprotein fractions using size exclusion chromatography prior to mass spectrometry. This associated these triacylglycerols with very low-density lipoprotein particles, and hence release of triacylglycerols into the blood from the liver. To test further the hepatic origin of these triacylglycerols we induced de novo lipogenesis in the mouse, comparing ob/ob and wild-type mice on a chow or high fat diet, confirming that de novo lipogenesis induced an increase in relatively shorter, more saturated fatty acids. CONCLUSIONS: Overall, these studies highlight hepatic de novo lipogenesis in the pathogenesis of metabolic dyslipidaemia in states where energy intake exceeds the capacity of adipose tissue

Hematopoietic IKBKE limits the chronicity of inflammasome priming and metaflammation

Obesity increases the risk of developing life-threatening metabolic diseases including cardiovascular disease, fatty liver disease, diabetes, and cancer. Efforts to curb the global obesity epidemic and its impact have proven unsuccessful in part by a limited understanding of these chronic progressive diseases. It is clear that low-grade chronic inflammation, or metaflammation, underlies the pathogenesis of obesity-associated type 2 diabetes and atherosclerosis. However, the mechanisms that maintain chronicity and prevent inflammatory resolution are poorly understood. Here, we show that inhibitor of κB kinase epsilon (IKBKE) is a novel regulator that limits chronic inflammation during metabolic disease and atherosclerosis. The pathogenic relevance of IKBKE was indicated by the colocalization with macrophages in human and murine tissues and in atherosclerotic plaques. Genetic ablation of IKBKE resulted in enhanced and prolonged priming of the NLRP3 inflammasome in cultured macrophages, in hypertrophic adipose tissue, and in livers of hypercholesterolemic mice. This altered profile associated with enhanced acute phase response, deregulated cholesterol metabolism, and steatoheptatitis. Restoring IKBKE only in hematopoietic cells was sufficient to reverse elevated inflammasome priming and these metabolic features. In advanced atherosclerotic plaques, loss of IKBKE and hematopoietic cell restoration altered plaque composition. These studies reveal a new role for hematopoietic IKBKE: to limit inflammasome priming and metaflammation

Community Code Verification Exercise for Simulating Sequences of Earthquakes and Aseismic Slip (SEAS)

Numerical simulations of sequences of earthquakes and aseismic slip (SEAS) have made great progress over past decades to address important questions in earthquake physics. However, significant challenges in SEAS modeling remain in resolving multiscale interactions between earthquake nucleation, dynamic rupture, and aseismic slip, and understanding physical factors controlling observables such as seismicity and ground deformation. The increasing complexity of SEAS modeling calls for extensive efforts to verify codes and advance these simulations with rigor, reproducibility, and broadened impact. In 2018, we initiated a community code‐verification exercise for SEAS simulations, supported by the Southern California Earthquake Center. Here, we report the findings from our first two benchmark problems (BP1 and BP2), designed to verify different computational methods in solving a mathematically well‐defined, basic faulting problem. We consider a 2D antiplane problem, with a 1D planar vertical strike‐slip fault obeying rate‐and‐state friction, embedded in a 2D homogeneous, linear elastic half‐space. Sequences of quasi‐dynamic earthquakes with periodic occurrences (BP1) or bimodal sizes (BP2) and their interactions with aseismic slip are simulated. The comparison of results from 11 groups using different numerical methods show excellent agreements in long‐term and coseismic fault behavior. In BP1, we found that truncated domain boundaries influence interseismic stressing, earthquake recurrence, and coseismic rupture, and that model agreement is only achieved with sufficiently large domain sizes. In BP2, we found that complexity of fault behavior depends on how well physical length scales related to spontaneous nucleation and rupture propagation are resolved. Poor numerical resolution can result in artificial complexity, impacting simulation results that are of potential interest for characterizing seismic hazard such as earthquake size distributions, moment release, and recurrence times. These results inform the development of more advanced SEAS models, contributing to our further understanding of earthquake system dynamics

Characterization of Human DNA Polymerase Delta and Its Subassemblies Reconstituted by Expression in the Multibac System

Mammalian DNA polymerase δ (Pol δ), a four-subunit enzyme, plays a crucial and versatile role in DNA replication and DNA repair processes. We have reconstituted human Pol δ complexes in insect cells infected with a single baculovirus into which one or more subunits were assembled. This system allowed for the efficient expression of the tetrameric Pol δ holoenzyme, the p125/p50 core dimer, the core+p68 trimer and the core+p12 trimer, as well as the p125 catalytic subunit. These were isolated in milligram amounts with reproducible purity and specific activities by a highly standardized protocol. We have systematically compared their activities in order to gain insights into the roles of the p12 and p68 subunits, as well as their responses to PCNA. The relative specific activities (apparent kcat) of the Pol δ holoenzyme, core+p68, core+p12 and p125/p50 core were 100, 109, 40, and 29. The corresponding apparent Kd's for PCNA were 7.1, 8.7, 9.3 and 73 nM. Our results support the hypothesis that Pol δ interacts with PCNA through multiple interactions, and that there may be a redundancy in binding interactions that may permit Pol δ to adopt flexible configurations with PCNA. The abilities of the Pol δ complexes to fully extend singly primed M13 DNA were examined. All the subassemblies except the core+p68 were defective in their abilities to completely extend the primer, showing that the p68 subunit has an important function in synthesis of long stretches of DNA in this assay. The core+p68 trimer could be reconstituted by addition of p12

The consequences of lipid remodelling of adipocyte membranes being functionally distinct from lipid storage in obesity

Obesity is a complex disorder where the genome interacts with diet and environmental factors to ultimately influence body mass, composition and shape. Numerous studies have investigated how bulk lipid metabolism of adipose tissue changes with obesity, and in particular how the composition of triglycerides (TGs) changes with increased adipocyte expansion. However, reflecting the analytical challenge posed by examining non-TG lipids in extracts dominated by TGs, the glycerophospholipid (PL) composition of cell membranes has been seldom investigated. PLs contribute to a variety of cellular processes including maintaining organelle functionality, providing an optimised environment for membrane-associated proteins and as pools for metabolites (e.g. choline for one-carbon metabolism and for methylation of DNA). We have conducted a comprehensive lipidomic study of white adipose tissue in mice who become obese either through genetic modification (ob/ob), diet (high fat diet) or a combination of the two using both solid phase extraction and ion mobility to increase coverage of the lipidome. Composition changes in seven classes of lipid (free fatty acids, diglycerides, TGs, phosphatidylcholines, lyso-phosphatidylcholines, phosphatidylethanolamines, and phosphatidylserines) correlated with perturbations in one-carbon metabolism and transcriptional changes in adipose tissue. We demonstrate that changes in TGs that dominate the overall lipid composition of white adipose tissue are distinct from diet-induced alterations of PLs, the predominant components of the cell membranes. PLs correlate better with transcriptional and one-carbon metabolism changes within the cell, suggesting the compositional changes that occur in cell membranes during adipocyte expansion have far-reaching functional consequences. Data is available at MetaboLights under the submission number: MTBLS1775

Production of Recombinant Human DNA Polymerase Delta in a Bombyx mori Bioreactor

Eukaryotic DNA polymerase δ (pol δ) plays a crucial role in chromosomal DNA replication and various DNA repair processes. It is thought to consist of p125, p66 (p68), p50 and p12 subunits. However, rigorous isolation of mammalian pol δ from natural sources has usually yielded two-subunit preparations containing only p125 and p50 polypeptides. While recombinant pol δ isolated from infected insect cells have some problems of consistency in the quality of the preparations, and the yields are much lower. To address these deficiencies, we have constructed recombinant BmNPV baculoviruses using MultiBac system. This method makes the generation of recombinant forms of pol δ containing mutations in any one of the subunits or combinations thereof extremely facile. From about 350 infected larvae, we obtained as much as 4 mg of pol δ four-subunit complex. Highly purified enzyme behaved like the one of native form by rigorous characterization and comparison of its activities on poly(dA)/oligo(dT) template-primer and singly primed M13 DNA, and its homogeneity on FPLC gel filtration. In vitro base excision repair (BER) assays showed that pol δ plays a significant role in uracil-intiated BER and is more likely to mediate LP BER, while the trimer lacking p12 is more likely to mediate SN BER. It seems likely that loss of p12 modulates the rate of SN BER and LP BER during the repair process. Thus, this work provides a simple, fast, reliable and economic way for the large-scale production of human DNA polymerase δ with a high activity and purity, setting up a new platform for our further research on the biochemical properties of pol δ, its regulation and the integration of its functions, and how alterations in pol δ function could contribute to the etiology of human cancer or other diseases that can result from loss of genomic stability

P50, the Small Subunit of DNA Polymerase Delta, Is Required for Mediation of the Interaction of Polymerase Delta Subassemblies with PCNA

Mammalian DNA polymerase δ (pol δ), a four-subunit enzyme, plays a crucial and versatile role in DNA replication and various DNA repair processes. Its function as a chromosomal DNA polymerase is dependent on the association with proliferating cell nuclear antigen (PCNA) which functions as a molecular sliding clamp. All four of the pol δ subunits (p125, p50, p68, and p12) have been reported to bind to PCNA. However, the identity of the subunit of pol δ that directly interacts with PCNA and is therefore primarily responsible for the processivity of the enzyme still remains controversial. Previous model for the network of protein-protein interactions of the pol δ-PCNA complex showed that pol δ might be able to interact with a single molecule of PCNA homotrimer through its three subunits, p125, p68, and p12 in which the p50 was not included in. Here, we have confirmed that the small subunit p50 of human pol δ truthfully interacts with PCNA by the use of far-Western analysis, quantitative ELISA assay, and subcellular co-localization. P50 is required for mediation of the interaction between pol δ subassemblies and PCNA homotrimer. Thus, pol δ interacts with PCNA via its four subunits

Genome-Wide Analysis of Protein-Protein Interactions and Involvement of Viral Proteins in SARS-CoV Replication

Analyses of viral protein-protein interactions are an important step to understand viral protein functions and their underlying molecular mechanisms. In this study, we adopted a mammalian two-hybrid system to screen the genome-wide intraviral protein-protein interactions of SARS coronavirus (SARS-CoV) and therefrom revealed a number of novel interactions which could be partly confirmed by in vitro biochemical assays. Three pairs of the interactions identified were detected in both directions: non-structural protein (nsp) 10 and nsp14, nsp10 and nsp16, and nsp7 and nsp8. The interactions between the multifunctional nsp10 and nsp14 or nsp16, which are the unique proteins found in the members of Nidovirales with large RNA genomes including coronaviruses and toroviruses, may have important implication for the mechanisms of replication/transcription complex assembly and functions of these viruses. Using a SARS-CoV replicon expressing a luciferase reporter under the control of a transcription regulating sequence, it has been shown that several viral proteins (N, X and SUD domains of nsp3, and nsp12) provided in trans stimulated the replicon reporter activity, indicating that these proteins may regulate coronavirus replication and transcription. Collectively, our findings provide a basis and platform for further characterization of the functions and mechanisms of coronavirus proteins