Fibrosis is a common pathology in many cardiac disorders and is driven by the activation of resident fibroblasts. The global posttranscriptional mechanisms underlying fibroblast-to-myofibroblast ...conversion in the heart have not been explored.
Genome-wide changes of RNA transcription and translation during human cardiac fibroblast activation were monitored with RNA sequencing and ribosome profiling. We then used RNA-binding protein-based analyses to identify translational regulators of fibrogenic genes. The integration with cardiac ribosome occupancy levels of 30 dilated cardiomyopathy patients demonstrates that these posttranscriptional mechanisms are also active in the diseased fibrotic human heart.
We generated nucleotide-resolution translatome data during the transforming growth factor β1-driven cellular transition of human cardiac fibroblasts to myofibroblasts. This identified dynamic changes of RNA transcription and translation at several time points during the fibrotic response, revealing transient and early-responder genes. Remarkably, about one-third of all changes in gene expression in activated fibroblasts are subject to translational regulation, and dynamic variation in ribosome occupancy affects protein abundance independent of RNA levels. Targets of RNA-binding proteins were strongly enriched in posttranscriptionally regulated genes, suggesting genes such as MBNL2 can act as translational activators or repressors. Ribosome occupancy in the hearts of patients with dilated cardiomyopathy suggested the same posttranscriptional regulatory network was underlying cardiac fibrosis. Key network hubs include RNA-binding proteins such as Pumilio RNA binding family member 2 (PUM2) and Quaking (QKI) that work in concert to regulate the translation of target transcripts in human diseased hearts. Furthermore, silencing of both PUM2 and QKI inhibits the transition of fibroblasts toward profibrotic myofibroblasts in response to transforming growth factor β1.
We reveal widespread translational effects of transforming growth factor β1 and define novel posttranscriptional regulatory networks that control the fibroblast-to-myofibroblast transition. These networks are active in human heart disease, and silencing of hub genes limits fibroblast activation. Our findings show the central importance of translational control in fibrosis and highlight novel pathogenic mechanisms in heart failure.
Plant-based emulsion gels can be used as solid animal fat substitutes for vegan sausages. For this reason, commercially available protein isolates with different amino acid profiles from pea, soy and ...potato (Pea-1, Pea-2, Soy, Potato) have been tested for their ability to form shape stable emulsions gels at neutral pH and upon heating to 72 °C. In order to obtain emulsion gels that are as solid as possible, the protein concentrations in the continuous phase (C
P
C, 8.0–11.5% (w/w)) and the oil mass fractions (65–80%) were varied. For leguminous proteins, a positive correlation of both parameters on emulsion rigidity was shown, indicating that both, interfacial and protein–protein interactions, are involved in structure reinforcement. Firmness increased with increasing content in cysteine (Pea-1 < Pea-2 < Soy) and the interactions were of electrostatic, hydrophobic and hydrophilic nature. Potato emulsion rigidity was independent of C
P
C and oil content. The emulsions showed a much higher degree in crosslinking, and very low charge density. Temperature-sweep analysis and CLSM revealed that Potato protein gelled as consequence to low temperature stability. Hence, the structure reinforcement in Potato emulsions mainly contributed to the protein network, with 70% oil and C
P
C 11.5% forming a hybrid gel with highest firmness. However, gelling of Potato protein also resulted in interfacial adsorption of protein aggregates and reduced interfacial stability with increasing C
P
C. This was demonstrated in the amount of extractable fat which was 2.0 and 0.6% for Pea-1 and 2 emulsions, 6.4% for Soy and 34.4% of total fat for Potato emulsions.
In hamburger manufacturing, meat is subjected to four main processing steps (pre-grinding, mixing, grinding, and forming), whereby muscle fibers are disintegrated. In this study, the influence of ...these process steps was characterized by structural (amount of non-intact cells (ANIC), CLS-Microscopy), functional (drip loss) and qualitative (soluble protein content, lactate dehydrogenase (LDH) activity, myoglobin content (Mb)) parameters of the meat. Therefore, meat samples were analyzed after each process step. Histological analyses revealed an increased ANIC with progressive processing. Thereby, the first and second grinding steps caused the strongest increases (factors 2.43 and 2.69). Comparable results were found in the relative LDH activity (factor 2.20 and 1.62) and the Mb concentration (factor 2.24 and 1.33) of the extracted meat solution. The findings suggest that the disintegration of the meat structure increases with progressive processing, causing more vulnerable structures which result in increased leakage of intramuscular substances. Further, the type of stress acting on the meat determines the extent of the changes. The presented findings enable manufacturers to precisely adjust their process towards more gentle production parameters and thus, to meet the legal regulations.
The large TSH-bound ectodomain of the thyrotropin receptor (TSHR) activates the transmembrane domain (TMD) indirectly via an internal agonist (IA). The ectodomain/TMD interface consists of a ...converging helix, a Cys-Cys-bridge-linked IA, and extracellular loops (ECL). To investigate the intramolecular course of molecular activation, especially details of the indirect activation, we narrowed down allosteric inhibition sites of negative allosteric modulator (NAM) by mutagenesis, homology modeling, and competition studies with positive allosteric modulator (PAM). From the inhibitory effects of NAM S37a on: 1) chimeras with swapped ectodomain, 2) stepwise N-terminal truncations, 3) distinct constitutively active mutations distributed across the hinge region and ECL, but not across the TMD, we conclude that S37a binds at the ectodomain/TMD interface, between the converging helix, ECL1, and the IA. This is also supported by the noncompetitive inhibition of PAM-C2-activation by S37a in the TSHR-TMD construct lacking the ectodomain. Mutagenesis studies on the IA and ECL were guided by our refined model of the ectodomain/TMD interface and indicate an interaction with the TSHR-specific residues E404 (preceding IA) and H478 (ECL1). At this new allosteric interaction site, NAM S37a blocks both TSH- and PAM-induced activation of the TSHR. Our refined models, mutations, and new allosteric binding pocket helped us to gain more detailed insights into the intramolecular course of TSHR activation at the ectodomain/TMD interface, including the delocalization of the converging helix and rearrangement of the conformation of IA. These changes are embedded between the ECL and cooperatively trigger active conformations of TMD. SIGNIFICANCE STATEMENT: The intramolecular activation mechanisms of the TSHR appear to be distinct from those of other G protein-coupled receptors, as the TSHR has a uniquely large N-terminal ectodomain that includes the hormone binding site and an internal agonist sequence. We present new molecular and structural insights into the interface between ectodomain and transmembrane domain in the TSHR, as well as the transfer of activation to the transmembrane domain. This knowledge is critical for understanding activation or inhibition of the receptor by allosteric ligands. We have identified a new allosteric antagonist binding pocket that is located exactly at this interface and possesses specific features that may allow the generation of potent highly TSHR-selective drugs, of potential value for the treatment of Graves' orbitopathy.
Little is known about the impact of trans-acting genetic variation on the rates with which proteins are synthesized by ribosomes. Here, we investigate the influence of such distant genetic loci on ...the efficiency of mRNA translation and define their contribution to the development of complex disease phenotypes within a panel of rat recombinant inbred lines.
We identify several tissue-specific master regulatory hotspots that each control the translation rates of multiple proteins. One of these loci is restricted to hypertrophic hearts, where it drives a translatome-wide and protein length-dependent change in translational efficiency, altering the stoichiometric translation rates of sarcomere proteins. Mechanistic dissection of this locus across multiple congenic lines points to a translation machinery defect, characterized by marked differences in polysome profiles and misregulation of the small nucleolar RNA SNORA48. Strikingly, from yeast to humans, we observe reproducible protein length-dependent shifts in translational efficiency as a conserved hallmark of translation machinery mutants, including those that cause ribosomopathies. Depending on the factor mutated, a pre-existing negative correlation between protein length and translation rates could either be enhanced or reduced, which we propose to result from mRNA-specific imbalances in canonical translation initiation and reinitiation rates.
We show that distant genetic control of mRNA translation is abundant in mammalian tissues, exemplified by a single genomic locus that triggers a translation-driven molecular mechanism. Our work illustrates the complexity through which genetic variation can drive phenotypic variability between individuals and thereby contribute to complex disease.
(1) Background: The selection of raw material and the postmortem processing of beef influence its quality, such as taste. In this study, the metabolome of beef from cows and heifers is examined for ...differences during aging. (2) Methods: Thirty strip loins from eight heifers and seven cows (breed code: 01–SBT) were cut into ten pieces and aged for 0, 7, 14, 21 and 28 days. Samples from the left strip loins were wet-aged in vacuum, while samples from right strip loins were dry-aged at 2 °C and 75% relative humidity. The beef samples were extracted with methanol–chloroform–water, and the polar fraction was used for 1H NMR analysis. (3) Results: The PCA and OPLS-DA showed that the metabolome of cows and heifers varied. Eight metabolites revealed significant differences (p < 0.05) in the samples from cows and heifers. The aging time and aging type of beef also affected the metabolome. Twenty-eight and 12 metabolites differed significantly (p < 0.05) with aging time and aging type, respectively. (4) Conclusions: The variations between cows and heifers and aging time affect the metabolome of beef. By comparison, the influence of aging type is present but less pronounced.
Larger processing equipment to produce minced meat could affect its structure due to intensive processing and a high energy intake in the meat mass. To assess if this would result in alterations in ...the minced meat quality, finely chopped meat (FCM) was added in different concentrations (15, 30, 45, 60, 75, 90, and 100%) to minced meat and quality parameters were analyzed. FCM was used to simulate different intensity of an unintended destruction of meat cells due to various processes. The amount of non-intact cells (ANIC) was determined histologically and furthermore, soluble protein content, water holding capacity, mechanical and sensory texture, and scanning electron and confocal laser scanning microscopy was applied to analyze the meat structure and quality. ANIC indicated that even adding 15% FCM was statistically (p < 0.05) distinguishable from 100% minced meat and 30% FCM had already 50 Vol.-% ANIC. In contrast, the addition of 15% or 30% FCM did not result in significant differences in drip loss of raw and cooked meat as well as mechanical and sensory texture analysis. This study showed that intensive processing might be detectable via ANIC, but that the minced meat quality was not affected.
The ban on piglet castration without anaesthesia poses a challenge for the meat industry since alternatives ensuring the production of flawless pork have to be established. The aim of this study was ...to evaluate the effects of biochar on skatole and indole concentration in faeces and plasma on a small scale in finishing boars to prove whether biochar was suitable for use in commercial pork production. Moreover, it was investigated whether biochar affects faecal properties or the performance. For a four-week trial period, 54 boars (bodyweight 97.2 ± 6.88 kg) were divided into three groups. The control (BC0) received no dietary biochar, one group received a diet containing 4% coated biochar (corresponding to 2% pure biochar) for the final two experimental weeks (BC2), and another group for the entire four weeks (BC4), respectively, prior to slaughter. Skatole and indole concentrations were measured in faeces and plasma at the beginning, in the middle and at the end of the trial. Mean skatole concentrations did not differ between groups, but in BC2 faecal skatole was significantly decreased at day 26, whereas in BC4 initial and final faecal skatole levels did not differ. At day 15 and 26, the faecal dry matter content was significantly higher in pigs fed the biochar diet (
< 0.05).
To reduce the risk of boar taint, intact male piglets are immuno- or surgically castrated. One alternative is reducing skatole by adding skatole reducing or adsorbing substances to the boars' diet. ...Charcoal with a high capacity for adsorbing skatole and indole in vitro (tested before, data not shown) was fed to the boars to test the hypothesis that a fat coating prevents the unspecific adsorption of charcoal before entry into the large intestine while increasing skatole adsorption. Twelve male and six female weaning piglets with initial body weights of 7.74 ± 0.75 kg were fed for 18 (or 19) days with either 2% pure (untreated) charcoal or 4% coated (50% charcoal + 50% fat-coating) charcoal or no charcoal. After euthanasia, skatole and indole were quantified in caecum and colon chyme. Skatole and indole contents in caecum chyme were significantly lower (
< 0.05) in the group fed with coated charcoal (33 ± 4.2, 7 ± 2.8 µg/g
, respectively) than in the group fed with pure charcoal (51 ± 7.3, 14 ± 3.0 µg/g
) or with no charcoal (73 ± 12.6, 15 ± 1.7 µg/g
). Similar effects were obvious for colon chyme. The results indicate that a fat coating of charcoal might prevent unspecific adsorption in the small intestine and might consequently lead to a higher adsorption capacity for skatole and indole in the large intestine, as skatole and indole concentrations in the chyme of caecum and colon were approximately 50% lower in the piglets who received coated charcoal.
The meat industry is typically using a mixture of fresh and frozen meat batters for minced meat production. Our goal was to find the exact threshold for fresh to frozen meat ratio capable of ...controlling the meat temperature during processing, but without having an adverse effect on the sensory quality of minced pork. To achieve this, the percentage of frozen meat used for the minced pork production was increased from 0% (control) to 50% (maximum) in 10% increments. To keep the minced meat temperature in control and make the processing resistant to fat smearing, the addition of 30% of frozen meat to the meat batter is sufficient. The soluble protein content, instrumental cutting force, and the sensory perceived firmness, juiciness, and inner cohesion were not affected by the addition of frozen meat. However, it has contributed to a significant increase of the drip loss and the amount of non-intact cells (ANIC). With the addition of frozen meat into the minced pork, the compliance to ANIC regulation by the German regulatory authorities is technologically (practically) almost impossible.
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