PCSK9 SNP rs11591147 is associated with low cholesterol levels but not with cognitive performance or noncardiovascular clinical events in an elderly population

Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) is a protein involved in LDL-cholesterol metabolism. The single-nucleotide polymorphism (SNP) rs11591147 has been associated with lower LDL-cholesterol and a lower risk of coronary heart disease. Because PCSK9 has high affinity to the LDL receptor, inhibiting PCSK9 is a testable therapeutic target for lipid-lowering therapy. Currently, several approaches to inhibit PCSK9 are under development, but it is unknown what the effects of those inhibitors will be on cognition or noncardiovascular clinical events. In this study, we assessed the association between rs11591147 and cognitive performance, activities of daily living (ADL), and noncardiovascular clinical events within 5,777 participants of the PROspective Study of Pravastatin in the Elderly at Risk (PROSPER). Rs11591147 was associated with 10% to 16% lower LDL cholesterol levels (P = 3.62 × 10⁻¹²), but was not associated with cognitive performance, ADL, or noncardiovascular clinical events in the PROSPER study. Our findings suggest that lower cholesterol levels due to genetic variation in the PCSK9 gene are not associated with cognitive performance, functional status, or noncardiovascular clinical events.     

In vivo effects of anacetrapib on preβ HDL: improvement in HDL remodeling without effects on cholesterol absorption

Cholesteryl ester transfer protein (CETP) transfers cholesteryl ester and triglyceride between HDL and apoB-containing lipoproteins. Anacetrapib (ANA), a reversible inhibitor of CETP, raises HDL cholesterol and lowers LDL cholesterol in dyslipidemic patients. We previously demonstrated that ANA increases macrophage-to-feces reverse cholesterol transport and fecal cholesterol excretion in hamsters, and increased preβ HDL-dependent cholesterol efflux via ABCA1 in vitro. However, the effects of ANA on in vivo preβ HDL have not been characterized. In vitro, ANA inhibited the formation of preβ, however in ANA-treated dyslipidemic hamsters, preβ HDL levels (measured by two-dimensional gel electrophoresis) were increased, in contrast to in vitro findings. Because changes in plasma preβ HDL have been proposed to potentially affect markers of cholesterol absorption with other CETP inhibitors, a dual stable isotope method was used to directly measure cholesterol absorption in hamsters. ANA treatment of hamsters (on either dyslipidemic or normal diet) had no effect on cholesterol absorption, while dalcetrapib-treated hamsters displayed an increase in cholesterol absorption. Taken together, these data support the notion that ANA promotes preβ HDL functionality in vivo, with no effects on cholesterol absorption.     

Sequence Analysis of In Vivo Defective Interfering-Like RNA of Influenza A H1N1 Pandemic Virus

Influenza virus defective interfering (DI) particles are naturally occurring noninfectious virions typically generated during in vitro serial passages in cell culture of the virus at a high multiplicity of infection. DI particles are recognized for the role they play in inhibiting viral replication and for the impact they have on the production of infectious virions. To date, influenza virus DI particles have been reported primarily as a phenomenon of cell culture and in experimentally infected embryonated chicken eggs. They have also been isolated from a respiratory infection of chickens. Using a sequencing approach, we characterize several subgenomic viral RNAs from human nasopharyngeal specimens infected with the influenza A(H1N1)pdm09 virus. The distribution of these in vivo-derived DI-like RNAs was similar to that of in vitro DIs, with the majority of the defective RNAs generated from the PB2 (segment 1) of the polymerase complex, followed by PB1 and PA. The lengths of the in vivo-derived DI-like segments also are similar to those of known in vitro DIs, and the in vivo-derived DI-like segments share internal deletions of the same segments. The presence of identical DI-like RNAs in patients linked by direct contact is compatible with transmission between them. The functional role of DI-like RNAs in natural infections remains to be established.     

Foot-and-Mouth Disease Virus Modulates Cellular Vimentin for Virus Survival

Foot-and-mouth disease virus (FMDV), the causative agent of foot-and-mouth disease, is an Aphthovirus within the Picornaviridae family. During infection with FMDV, several host cell membrane rearrangements occur to form sites of viral replication. FMDV protein 2C is part of the replication complex and thought to have multiple roles during virus replication. To better understand the role of 2C in the process of virus replication, we have been using a yeast two-hybrid approach to identify host proteins that interact with 2C. We recently reported that cellular Beclin1 is a natural ligand of 2C and that it is involved in the autophagy pathway, which was shown to be important for FMDV replication. Here, we report that cellular vimentin is also a specific host binding partner for 2C. The 2C-vimentin interaction was further confirmed by coimmunoprecipitation and immunofluorescence staining to occur in FMDV-infected cells. It was shown that upon infection a vimentin structure forms around 2C and that this structure is later resolved or disappears. Interestingly, overexpression of vimentin had no effect on virus replication; however, overexpression of a truncated dominant-negative form of vimentin resulted in a significant decrease in viral yield. Acrylamide, which causes disruption of vimentin filaments, also inhibited viral yield. Alanine scanning mutagenesis was used to map the specific amino acid residues in 2C critical for vimentin binding. Using reverse genetics, we identified 2C residues that are necessary for virus growth, suggesting that the interaction between FMDV 2C and cellular vimentin is essential for virus replication.     

Community composition, correlations among taxa, prevalence, and richness in gastrointestinal parasites of baboons in Senegal, West Africa

Studies of gastrointestinal parasite prevalence in Papio have either focused on a single troop or compared prevalence among troops that share migrants but differ in degree of human contact. Little is known about the extent of variation in prevalence where obvious factors that may drive prevalence (e.g., human contact) are absent, so it is difficult to interpret variation when these factors are present. To address this issue, we studied troops of Guinea baboons (Papio papio) that had almost no contact with humans or domesticated species of plants or animals. We tested the null hypotheses that community composition, richness, and prevalence would be similar between groups in two comparisons: (1) between troops in the same locality with no known differences in drivers of prevalence, and (2) between samples at the same location taken more than 20 years apart. We collected anonymous fecal samples from two troops of baboons living in a wilderness site, Mt. Assirik, in the Niokolo-Koba National Park, Republic of Senegal, West Africa. We collected samples from two valleys and analyzed prevalence and richness with respect to place and time. Both prevalence and richness were similar in the two valleys, but significant changes emerged in both prevalence and community composition compared with the previous survey in 1978–1979. We also found that the nematode Enterobius and a fluke, Watsonius, co-occurred within hosts more frequently than expected. This phenomenon has not been previously noted in the literature, and it suggests common environmental drivers or facilitation among these parasites.     

Predation of Alouatta puruensis by Boa constrictor

Reports of successful predator attacks on primates are rare. Primates from all major radiations are particularly susceptible to raptors, carnivores, and snakes. Among New World primates, reports of snake predation are limited to medium- and small-bodied species. Here, we report the first documented case of successful predation of an atelid by a snake—an adult female Purús red howler monkey, Alouatta puruensis, that was subdued by a ca. 2-m-long Boa constrictor in an arboreal setting at a height of 7.5 m above the ground. The victim belonged to a group composed of six individuals (one adult male, two adult females, two juveniles, and one infant) that inhabited a ca. 2.5-ha forest fragment in the State of Rondônia, western Brazilian Amazon. The boa applied the species’ typical hunting behavior of striking and immediately coiling around its prey and then killing it through constriction (probably in less than 5 min), but the entire restraint period lasted 38 min. The attack occurred around noon. The howler was swallowed head-first in 76 min. The only group member to respond to the distress vocalization emitted by the victim was the other adult female, which was closest to the location where the attack occurred. This female ran toward the snake, also vocalizing, and hit it with her hands several times, but the snake did not react and she moved off to a nearby tree from where she watched most of the interaction. The remaining group members stayed resting at a height approximately 15 m above the victim in a nearby tree without showing any overt signs of stress, except for a single whimper vocalization. This event indicates that even large-bodied atelids are vulnerable to predation by large snakes and suggests that B. constrictor may be a more common predator of primates.     

Elucidation of the Structure and Reaction Mechanism of Sorghum Hydroxycinnamoyltransferase and Its Structural Relationship to Other Coenzyme A-Dependent Transferases and Synthases

Hydroxycinnamoyltransferase (HCT) from sorghum (Sorghum bicolor) participates in an early step of the phenylpropanoid pathway, exchanging coenzyme A (CoA) esterified to p-coumaric acid with shikimic or quinic acid as intermediates in the biosynthesis of the monolignols coniferyl alcohol and sinapyl alcohol. In order to elucidate the mode of action of this enzyme, we have determined the crystal structures of SbHCT in its apo-form and ternary complex with shikimate and p-coumaroyl-CoA, which was converted to its product during crystal soaking. The structure revealed the roles of threonine-36, serine-38, tyrosine-40, histidine-162, arginine-371, and threonine-384 in catalysis and specificity. Based on the exact chemistry of p-coumaroyl-CoA and shikimic acid in the active site and an analysis of kinetic and thermodynamic data of the wild type and mutants, we propose a role for histidine-162 and threonine-36 in the catalytic mechanism of HCT. Considering the calorimetric data, substrate binding of SbHCT should occur sequentially, with p-coumaroyl-CoA binding prior to the acyl acceptor molecule. While some HCTs can use both shikimate and quinate as an acyl acceptor, SbHCT displays low activity toward quinate. Comparison of the structure of sorghum HCT with the HCT involved in chlorogenic acid synthesis in coffee (Coffea canephora) revealed many shared features. Taken together, these observations explain how CoA-dependent transferases with similar structural features can participate in different biochemical pathways across species.Lignin is a major structural and protective component of plant cell walls. Lignin exists as a polymer of mainly three hydroxycinnamyl alcohols and related compounds, referred to as monolignols. The most common monolignols are coniferyl, sinapyl, and p-coumaryl alcohol (Ralph et al., 2004; Vanholme et al., 2010). After polymerization, structures derived from those compounds are referred to as guaiacyl, syringyl, and p-hydroxyphenyl subunits, respectively. The specific composition of lignin subunits varies among species, tissues, and developmental stages. Gymnosperm trees produce lignin that is primarily made of guaiacyl subunits, angiosperm trees contain guaiacyl and syringyl subunits, whereas grasses contain guaiacyl and syringyl subunits with small amounts (approximately 5%) of p-hydroxyphenyl residues. This observed variation in subunit composition across species may reflect the heterogeneity in substrate specificity and kinetic parameters among various monolignol biosynthetic enzymes (Weng et al., 2008).Biosynthesis of the monolignols occurs via the phenylpropanoid pathway using Phe precursors (Vanholme et al., 2010). Phe ammonia lyase, cinnamate-4-hydroxylase, and 4-coumarate coenzyme A (CoA) ligase (4CL) generate p-coumaroyl-CoA from Phe (Vanholme et al., 2010). Grasses can bypass cinnamate-4-hydroxylase by using Tyr as a substrate for Phe ammonia lyase (Neish, 1961; Rösler et al., 1997). The hydroxycinnamoyltransferase (HCT) enzymes exchange the CoA functionality esterified to p-coumaric acid with shikimic or quinic acid to allow for the subsequent conversion of the p-coumaroyl moiety to a caffeoyl moiety by p-coumarate-3′-hydroxylase (C3′H). The hydroxycinnamoyl-CoA shikimate hydroxycinnamoyltransferases (HSTs) exhibit preference for shikimate, whereas the hydroxycinnamoyl-CoA quinate hydroxycinnamoyltransferases prefer quinate as a substrate (Sander and Petersen, 2011). Subsequent reactions ultimately lead to coniferyl and sinapyl alcohol via reduction of the γ-carbon on the propane side chain and substitution of the C3 and C5 positions of the phenol ring (Boerjan et al., 2003).Sorghum (Sorghum bicolor) is an attractive bioenergy crop with typical dry biomass yields between 20 and 25 Mg ha⁻¹ and yields as high as 40 Mg ha⁻¹ possible under optimal conditions (Venuto and Kindiger, 2008). Moreover, sorghum utilizes nitrogen-based fertilizer more efficiently than maize (Zea mays) and sugarcane (Saccharum officinarum), leading to less groundwater contamination and lower CO₂ emission (Propheter and Staggenborg, 2010; Wortmann and Regassa, 2011). Overall, sorghum has a higher sugar yield potential per land area and requires less water for growth than maize, allowing it to grow in a more diverse range of environments (Saballos, 2008). The sorghum genome sequence has been released (Paterson et al., 2009), and Targeting Induced Local Lesions in Genomes populations exist (Xin et al., 2008) in which various cell wall mutants have been identified (Sattler et al., 2012; Vermerris and Saballos, 2012).A detailed understanding of the catalytic mechanism of phenylpropanoid-related enzymes will enable the targeted modification of lignin subunit composition. The presence of lignin poses a major obstacle to the production of biofuels and chemicals from lignocellulosic biomass, because of its ability to hinder the activity of enzymes required to degrade cellulose to sugars that can be fermented for ethanol production (Yang and Wyman, 2004; Berlin et al., 2006). Genetic modification of plant cell wall composition, especially lignin content and subunit composition, has been shown to improve biomass conversion to fermentable sugars (Chen and Dixon, 2007; Vermerris et al., 2007; Jung et al., 2012). In particular, HCT silencing in Arabidopsis (Arabidopsis thaliana) causes an accumulation of p-hydroxyphenyl residues in the lignin and decreased content of guaiacyl and syringyl residues, leading to a dwarf phenotype (Li et al., 2010). Down-regulation of HCT has also been shown to result in decreased plant growth in alfalfa (Medicago sativa; Shadle et al., 2007). Concomitantly, ruminant digestibility and the yield of fermentable sugars following enzymatic saccharification increased (Chen and Dixon, 2007; Shadle et al., 2007). Reduced HCT activity may alter cell wall polymer interactions and allow better access of cellulolytic enzymes to the cellulose. Therefore, it has the potential to reduce the energy and processing costs associated with the conversion of biomass to fuels and chemicals. However fine-tuning will be necessary to limit the negative impacts on plant growth, which will require a detailed understanding of the catalytic mechanism of HCT.Given the difference in lignin subunit composition among different species and the prominence of grasses among dedicated bioenergy crops, we have focused on elucidating the crystal structure and activity of monolignol-related enzymes of sorghum, starting with the HST-like HCT. HCT belongs to the BAHD superfamily of plant-specific acyl-CoA-dependent acyltransferases (Ma et al., 2005; D’Auria, 2006). However, the BAHD superfamily has functionally and structurally diverse members that frequently possess little (as low as 10%) sequence identity among them (St-Pierre and Luca, 2000). Recent studies led to the crystal structure of the HST-like HCT from robusta coffee (Coffea canephora), an angiosperm dicot with a binding pocket elucidated by molecular docking and mutagenesis (Lallemand et al., 2012). In this report, we present the three-dimensional structures of HCT in its apo-form and ternary complex, supplemented by mutagenic studies to elucidate its reaction mechanism and structural relationship to other members in this growing functional class.