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Ethylene regulates fruit ripening and several plant functions (germination, plant growth, plant-microbe interactions). Protein quantification of ethylene receptors (ETRs) is essential to study their functions, but is impaired by low resolution tools such as antibodies that are mostly nonspecific, or the lack of sensitivity of shotgun proteomic approaches. We developed a targeted proteomic method, to quantify low-abundance proteins such as ETRs, and coupled this to mRNAs analyses, in two tomato lines: Wild Type (WT) and Never-Ripe (NR) which is insensitive to ethylene because of a gain-of-function mutation in ETR3. We obtained mRNA and protein abundance profiles for each ETR over the fruit development period. Despite a limiting number of replicates, we propose Pearson correlations between mRNA and protein profiles as interesting indicators to discriminate the two genotypes: such correlations are mostly positive in the WT and are affected by the NR mutation. The influence of putative post-transcriptional and post-translational changes are discussed. In NR fruits, the observed accumulation of the mutated ETR3 protein between ripening stages (Mature Green and Breaker + 8 days) may be a cause of NR tomatoes to stay orange. The label-free quantitative proteomics analysis of membrane proteins, concomitant to Parallel Reaction Monitoring analysis, may be a resource to study changes over tomato fruit development. These results could lead to studies about ETR subfunctions and interconnections over fruit development. Variations of RNA-protein correlations may open new fields of research in ETR regulation. Finally, similar approaches may be developed to study ETRs in whole plant development and plant-microorganism interactions.

The control of growth and development of all living organisms is a complex and dynamic process that requires the harmonious expression of numerous genes. Gene expression is mainly controlled by the activity of sequence-specific DNA binding proteins called transcription factors (TFs). Amongst the various classes of eukaryotic TFs, the MYB superfamily is one of the largest and most diverse, and it has considerably expanded in the plant kingdom. R2R3-MYBs have been extensively studied over the last 15 years. However, DNA-binding specificity has been characterized for only a small subset of these proteins. Therefore, one of the remaining challenges is the exhaustive characterization of the DNA-binding specificity of all R2R3-MYB proteins. In this study, we have developed a library of Arabidopsis thaliana R2R3-MYB open reading frames, whose DNA-binding activities were assayed in vivo (yeast one-hybrid experiments) with a pool of selected cis-regulatory elements. Altogether 1904 interactions were assayed leading to the discovery of specific patterns of interactions between the various R2R3-MYB subgroups and their DNA target sequences and to the identification of key features that govern these interactions. The present work provides a comprehensive in vivo analysis of R2R3-MYB binding activities that should help in predicting new DNA motifs and identifying new putative target genes for each member of this very large family of TFs. In a broader perspective, the generated data will help to better understand how TF interact with their target DNA sequences.

Abstract Background Almonds have been shown to lower LDL cholesterol but there is limited information regarding their effects on the dyslipidemia characterized by increased levels of very low density lipoproteins (VLDL) and small, dense low-density lipoprotein (LDL) particles that is associated with abdominal adiposity and high carbohydrate intake. The objective of the present study was to test whether substitution of almonds for other foods attenuates carbohydrate-induced increases in small, dense LDL in individuals with increased abdominal adiposity. Methods This was a randomized cross-over study of three 3wk diets, separated by 2wk washouts: a higher-carbohydrate (CHO) reference diet (CHOhigh), a higher-CHO diet with isocaloric substitution of 20% kcal (E) from almonds (CHOhigh + almonds), and a lower-CHO reference diet (CHOlow) in 9 men and 15 women who were overweight or obese. The two CHOhigh diets contained 50% carbohydrate, 15% protein, 35% fat (6% saturated, 21% monounsaturated, 8% polyunsaturated), while the CHOlow diet contained 25% carbohydrate, 28% protein, 47% fat (8% saturated, 28% monounsaturated, 8% polyunsaturated). Lipoprotein subfraction concentrations were measured by ion mobility. Results Relative to the CHOlow diet: 1) the CHOhigh + almonds diet significantly increased small, dense LDLIIIa (mean difference ± SE: 28.6 ± 10.4 nmol/L, P = 0.008), and reduced LDL-peak diameter (− 1.7 ± 0.6 Å, P = 0.008); 2) the CHOhigh diet significantly increased medium-sized LDLIIb (24.8 ± 11.4 nmol/L, P = 0.04) and large VLDL (3.7 ± 1.8 nmol/L, P = 0.05). Relative to CHOlow, the effects of CHOhigh on LDLIIIa (17.7 ± 10.6 nmol/L) and LDL-peak diameter (− 1.1 ± 0.6 Å) were consistent with those of CHOhigh + almonds, and the effects of CHOhigh + almonds on LDLIIb (21.0 ± 11.2 nmol/L) and large VLDL (2.8 ± 1.8 nmol/L) were consistent with those of CHOhigh, but did not achieve statistical significance (P > 0.05). None of the variables examined showed a significant difference between the CHOhigh + almonds and CHOhigh diets (P > 0.05). Conclusion Our analyses provided no evidence that deriving 20% E from almonds significantly modifies increases in levels of small, dense LDL or other plasma lipoprotein changes induced by a higher carbohydrate low saturated fat diet in individuals with increased abdominal adiposity. Trial registration Clinicaltrials.gov NCT01792648.

ABSTRACT Interindividual variation in the composition of the human gut microbiome was examined in relation to demographic and anthropometric traits, and to changes in dietary saturated fat intake and protein source. One hundred nine healthy men and women aged 21 to 65, with BMIs of 18 to 36, were randomized, after a two-week baseline diet, to high (15% total energy [E])- or low (7%E)-saturated-fat groups and randomly received three diets (four weeks each) in which the protein source (25%E) was mainly red meat (beef, pork) (12%E), white meat (chicken, turkey) (12%E), and nonmeat sources (nuts, beans, soy) (16%E). Taxonomic characterization using 16S ribosomal DNA was performed on fecal samples collected at each diet completion. Interindividual differences in age, body fat (%), height, ethnicity, sex, and alpha diversity (Shannon) were all significant factors, and most samples clustered by participant in the PCoA ordination. The dietary interventions did not significantly alter the overall microbiome community in ordination space, but there was an effect on taxon abundance levels. Saturated fat had a greater effect than protein source on taxon differential abundance, but protein source had a significant effect once the fat influence was removed. Higher alpha diversity predicted lower beta diversity between the experimental and baseline diets, indicating greater resistance to change in people with higher microbiome diversity. Our results suggest that interindividual differences outweighed the influence of these specific dietary changes on the microbiome and that moderate changes in saturated fat level and protein source correspond to modest changes in the microbiome. IMPORTANCE The microbiome has proven to influence health and disease, but how combinations of external factors affect the microbiome is relatively unknown. Diet can cause changes, but this is usually achieved by altering macronutrient ratios and has not focused on dietary protein source or saturated fat intake levels. In addition, each individual’s unique microbiome profile can be an important factor during studies, and it has even been shown to affect therapeutic outcomes. We show here that the effects of individual differences outweighed the effect of experimental diets and that protein source is less influential than saturated fat level. This suggests that fat and protein composition, separate from macronutrient ratio and carbohydrate composition, is an important consideration in dietary studies.

ABSTRACT Diet can influence the composition of the human microbiome, and yet relatively few dietary ingredients have been systematically investigated with respect to their impact on the functional potential of the microbiome. Dietary resistant starch (RS) has been shown to have health benefits, but we lack a mechanistic understanding of the metabolic processes that occur in the gut during digestion of RS. Here, we collected samples during a dietary crossover study with diets containing large or small amounts of RS. We determined the impact of RS on the gut microbiome and metabolic pathways in the gut, using a combination of “omics” approaches, including 16S rRNA gene sequencing, metaproteomics, and metabolomics. This multiomics approach captured changes in the abundance of specific bacterial species, proteins, and metabolites after a diet high in resistant starch (HRS), providing key insights into the influence of dietary interventions on the gut microbiome. The combined data showed that a high-RS diet caused an increase in the ratio of Firmicutes to Bacteroidetes, including increases in relative abundances of some specific members of the Firmicutes and concurrent increases in enzymatic pathways and metabolites involved in lipid metabolism in the gut. IMPORTANCE This work was undertaken to obtain a mechanistic understanding of the complex interplay between diet and the microorganisms residing in the intestine. Although it is known that gut microbes play a key role in digestion of the food that we consume, the specific contributions of different microorganisms are not well understood. In addition, the metabolic pathways and resultant products of metabolism during digestion are highly complex. To address these knowledge gaps, we used a combination of molecular approaches to determine the identities of the microorganisms in the gut during digestion of dietary starch as well as the metabolic pathways that they carry out. Together, these data provide a more complete picture of the function of the gut microbiome in digestion, including links between an RS diet and lipid metabolism and novel linkages between specific gut microbes and their metabolites and proteins produced in the gut.

The aim of this study was to investigate the extent to which inflammation is linked with plasma endothelial lipase (EL) concentrations among healthy sedentary men. Plasma C-reactive protein (CRP) concentrations were measured with a highly sensitive commercial immunoassay, plasma interleukin-6 (IL-6) concentrations were measured using a commercial ELISA, and plasma secretory phospholipase A2 type IIA (sPLA2-IIA) concentrations were measured using a commercial assay in a sample of 74 moderately obese men (mean body mass index, 29.8 ± 5.2 kg/m2). Plasma EL concentrations were positively correlated with various indices of obesity, fasting plasma insulin, and plasma CRP, IL-6, and sPLA2-IIA concentrations. Multiple regression analyses revealed that plasma CRP concentrations explained 14.5% (P = 0.0008) of the variance in EL concentrations. When entered into the model, LPL activity accounted for 16.1% (P < 0.0001) and plasma CRP concentrations accounted for 20.9% (P < 0.0001) of the variance in EL concentrations. The combined impact of visceral adipose tissue (VAT) and of an inflammation score on EL concentrations was investigated. Among subjects with high or low VAT, those having a high inflammation score based on plasma CRP, IL-6, and sPLA2-IIA concentrations had increased plasma EL concentrations (P = 0.0005). In conclusion, our data reveal a strong association between proinflammatory cytokines and plasma EL concentrations among healthy people with low or high VAT levels.

Apolipoprotein C-III (apoC-III) production rate (PR) is strongly correlated with plasma triglyceride (TG) levels. ApoC-III exists in three different isoforms, according to the sialylation degree of the protein. We investigated the kinetics and respective role of each apoC-III isoform in modulating intravascular lipid/lipoprotein metabolism. ApoC-III kinetics were measured in a sample of 18 healthy men [mean age (±SD) 42.1 ± 9.5 years, body mass index 29.8 ± 4.6 kg/m2] using a primed-constant infusion of l-(5,5,5-D3) leucine for 12 h. Mono-sialylated and di-sialylated apoC-III (apo-CIII1 and apoC-III2) exhibited similar PRs (means ± SD, 1.22 ± 0.49 mg/kg/day vs. 1.15 ± 0.59 mg/kg/day, respectively) and similar fractional catabolic rates (FCRs) (0.51 ± 0.13 pool/day vs. 0.61 ± 0.24 pool/day, respectively). Nonsialylated apoC-III (apoC-III0) had an 80% lower PR (0.25 ± 0.12 mg/kg/day) and a 60% lower FCR (0.21 ± 0.07 pool/day) (P < 0.0001 for comparison with CIII1 and CIII2 isoforms). The PRs of apoC-III1 and apoC-III2 were more strongly correlated with plasma TG levels (r > 0.8, P < 0.0001) than was apoC-III0 PR (r = 0.54, P < 0.05). Finally, the PR of apoC-III2 was strongly correlated with the proportion of LDL

The impact of a low-fat diet and a high-MUFA diet on apolipoprotein A-I (apoA-I), apoA-II, and VLDL-apoB-100 metabolism in conditions of unrestricted (ad libitum) energy intake was compared in 65 men randomly assigned to one of two predefined experimental diets. A subsample of 18 men participated in the kinetic study. Before and after the 6–7 week dietary intervention, kinetic subjects received a primed-constant infusion of [5,5,5-2H3]l-leucine for 12 h under feeding conditions. ApoA-I production rate (PR; −31.5%; P < 0.001) and fractional catabolic rate (FCR; −24.3%; P < 0.05) were significantly decreased after the low-fat diet. These changes in apoA-I PR and FCR with the low-fat diet were also significantly different from those observed with the high-MUFA diet (P < 0.01 and P < 0.05, respectively). ApoA-II FCR was significantly increased in the high-MUFA group only. No significant within- or between-diet difference was found in VLDL-apoB-100 PR or FCR.These results emphasize the differential impact of the low-fat diet and high-MUFA diet on HDL metabolism.

Mice lacking hepatic lipase have been reported to express mild hyperlipidemia characterized by increased concentrations of large high density lipoproteins, but normal concentrations of lipoproteins containing apolipoprotein B. Whereas hepatic lipase has been implicated in the clearance and processing of chylomicron remnants in rats, no such defect was found in these mice. We have further characterized the abnormal lipoprotein phenotype in young hepatic lipase-deficient mice and have found more pronounced elevations of high density lipoproteins associated in particular with a 5-fold increase in plasma concentrations of apolipoprotein E. In addition, there was a reduction in the concentration of low density lipoproteins containing apolipoprotein B-100 and B-48 relative to precursor lipoproteins of lower density and a pronounced deficiency of apolipoprotein B-containing low density lipoproteins with density exceeding 1.029 g/mL. Conversion of radiolabeled rabbit intermediate density lipoproteins to low density lipoproteins was reduced by 6-fold as compared with wild-type mice. Although clearance of cholesteryl ester-labeled chylomicrons from the blood was unimpaired in the deficient mice, that of chylomicron remnants was reduced. Furthermore, endocytosis of chylomicron cholesteryl esters into liver cells occurred more rapidly than in wild-type mice. The unimpaired hepatic clearance of injected chylomicron particles in hepatic lipase-deficient mice may be the result of greater acquisition of apoE from high density lipoproteins during remnant formation. These studies thus demonstrate a critical role for mouse hepatic lipase in the formation of small, dense low density lipoproteins, as well as participation in the normal clearance and processing of chylomicron remnants.—Qiu, S., N. Bergeron, L. Kotite, R. M. Krauss, A. Bensadoun, and R. J. Havel. Metabolism of lipoproteins containing apolipoprotein B in hepatic lipase-deficient mice.