Metabolic and microbial analyses of the surface and inner part of wet-aged and dry-aged beef.

The effects of aging and microbial growth on the metabolome of aged beef were investigated in this study. The metabolome of beef is influenced by the aging method applied. This includes the aging-related changes in metabolism and the presence of microorganisms on the beef during aging that may affect the beef and its quality. The inner part and the trimmed surface of dry-aged (the surface of dry-aged beef is also called the "crust" due to its drying during aging) and wet-aged beef were analyzed by 1 H nuclear magnetic resonance (NMR) spectroscopy over aging periods up to 28 days at intervals of 7 days, and the former also by microbiological analysis. The metabolome detected by 1 H NMR spectroscopy demonstrated changes over the aging time of beef and differed depending on the sampling location (surface or inner part of beef). The influence of the microbiota on changes in the metabolome can be negligible due to the low microbial growth on the surface of dry-aged beef (<3 log CFU/g). Therefore, the aging-related metabolism postmortem of the analyzed dry-aged beef might be the main factor for metabolic changes. The significantly (p < 0.05) higher amino acids and inosine concentrations and lower inosine 5'-monophosphate concentrations suggested enhanced protein degradation and energy metabolism in the wet-aged beef compared to the dry-aged beef, probably due to the combined influence of the aging and the microbiota on the wet-aged beef and, thus, its metabolic changes.

A review on the relation between grinding process and quality of ground meat.

This review is providing an overview of the actual and past research in the field of ground meat. The forces that are acting in the meat grinder are well understood. The higher the forces that are acting on the meat while grinding, the stronger the disintegration of the meat cells after the process. These forces can be calculated as energy transfer in meat grinders using specific mechanical energy (SME). The amount of non-intact cells (ANIC) can be used to describe the extent of disintegrated cells. Different methods are available to rate the quality of ground meat, which is mainly influenced by the raw material and processing. Over the past decades of industrialization, the landscape of ground meat production has changed. However, the effects of the process adjustments on the quality of ground meat are not yet sufficiently described in the literature.

Variations in the Metabolome of Unaged and Aged Beef from Black-and-White Cows and Heifers by 1H NMR Spectroscopy.

(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.

Metabolic, proteomic and microbial changes postmortem and during beef aging.

The purpose of this review is to provide an overview of the current knowledge about proteomic and metabolic changes in beef, the microbiological alteration postmortem and during aging, and observe the influence on beef quality parameters, such as tenderness, taste and flavor. This review will also focus on the different aging types (wet- and dry-aging), the aging or postmortem time of beef and their effect on the proteome and metabolome of beef. The Ca2+ homeostasis and adenosine 5'-triphosphate breakdown are the main reactions in the pre-rigor phase. After rigor mortis, the enzymatic degradation of connective tissues and breakdown of energy metabolism dominate molecular changes in beef. Important metabolic processes leading to the formation of saccharides, nucleotides, organic acids (e.g. lactic acid), creatine and fatty acids are considered in this context as possible flavor precursors or formers of beef flavor and taste. Flavor precursors are substrates for lipid oxidation, Strecker degradation and Maillard reaction during cooking or roasting. The findings presented should serve as a basis for a better understanding of beef aging and its molecular effects and are intended to contribute to meeting the challenges of improving beef quality.

Effect of sampling position in fresh, dry-aged and wet-aged beef from M. Longissimus dorsi of Simmental cattle analyzed by 1H NMR spectroscopy.

The aging of beef affects the metabolome and, thus, its quality, such as taste or tenderness. In addition to the aging method, intrinsic factors, such as breed, feed and muscle type, also have an effect on beef's metabolome. It is not known yet whether the position of the sampling in large muscles also has an influence on beef's metabolome and its aging outcome. The effect of the sampling position in M. longissimus dorsi as a large muscle was investigated in dry-aged and wet-aged beef over an aging period of 28 days. In this study, we analyzed 360 samples out of the entire length of M. longissimus dorsi of 18 'Simmental' young bulls by 1H NMR spectroscopy. The position in the muscle affected the polar fraction of metabolome of non-aged and aged beef significantly. However, sampling position did not overlay significant differences in the metabolome of dry-aged and wet-aged beef. The aging time of beef also had a significant effect on the metabolome. Marker metabolites, such as leucine, isoleucine, inosine 5'-monophosphate and hypoxanthine, were found to be indicative of the aging time applied. In addition, marker metabolites (lactic acid, anserine, O-acetyl-L-carnitine) were identified for the aging type applied.

Preliminary Test of the Reduction Capacity for the Intestinal Adsorption of Skatole and Indole in Weaning Piglets by Pure and Coated Charcoal.

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 (p < 0.05) in the group fed with coated charcoal (33 ± 4.2, 7 ± 2.8 µg/gDM, respectively) than in the group fed with pure charcoal (51 ± 7.3, 14 ± 3.0 µg/gDM) or with no charcoal (73 ± 12.6, 15 ± 1.7 µg/gDM). 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.

Identification of disease-relevant modulators of the SHH pathway in the developing brain.

Pathogenic gene variants in humans that affect the sonic hedgehog (SHH) pathway lead to severe brain malformations with variable penetrance due to unknown modifier genes. To identify such modifiers, we established novel congenic mouse models. LRP2-deficient C57BL/6N mice suffer from heart outflow tract defects and holoprosencephaly caused by impaired SHH activity. These defects are fully rescued on a FVB/N background, indicating a strong influence of modifier genes. Applying comparative transcriptomics, we identified Pttg1 and Ulk4 as candidate modifiers upregulated in the rescue strain. Functional analyses showed that ULK4 and PTTG1, both microtubule-associated proteins, are positive regulators of SHH signaling, rendering the pathway more resilient to disturbances. In addition, we characterized ULK4 and PTTG1 as previously unidentified components of primary cilia in the neuroepithelium. The identification of genes that powerfully modulate the penetrance of genetic disturbances affecting the brain and heart is likely relevant to understanding the variability in human congenital disorders.

A trans locus causes a ribosomopathy in hypertrophic hearts that affects mRNA translation in a protein length-dependent fashion.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Evaluation of Coated Biochar as an Intestinal Binding Agent for Skatole and Indole in Male Intact Finishing Pigs.

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 (p < 0.05).

Covid-19 pandemic effects on food safety - Multi-country survey study.

This study provides an important insight into the response of food safety systems during the first months of the pandemic, elevating the perspective of preventing Covid-19 within conventional food safety management systems. A multi-country survey was conducted in 16 countries involving 825 food companies. Based on the results of the survey, it is obvious that the level of maturity of a food safety system in place is the main trigger in classifying companies and their responses to the pandemic challenge. Staff awareness and hygiene are the two most important attributes in combating Covid-19, opposed to temperature checking of workers in food establishment and health protocols from the World Health Organization, recognized as attributes with limited salience and importance. Companies confirmed implementation of more restrictive hygiene procedures during the pandemic and the need for purchasing more additional personal protective equipment. Retailers were identified as the food supply chain link mostly affected by the pandemic opposed to food storage facilities ranked as least affected. During this challenging period, all companies declared that food safety has not been compromised at any moment. It is important to note that less than a half of the food companies had documented any emergency plans associated with pandemics and health issues in place.

Analysis of aging type- and aging time-related changes in the polar fraction of metabolome of beef by 1H NMR spectroscopy.

The tenderness and taste of beef is improved by either dry- or wet-aging or a combination of both. The objective was to develop a validated method for detecting differences in the polar fraction of metabolome in dry-aged and wet-aged beef over the aging time and quantifying the metabolites of interest by 1H NMR spectroscopy using beef. Sixty strip loin (M. longissimus dorsi) samples aged in different ways (wet-aging vs. dry-aging) and aging times (0, 7, 14, 21, 28 days) were analyzed. The aging type could be defined by linear discriminant analysis with an accuracy of 95%. Ten (lactic acid, alanine, methionine, fumaric acid, inosine, inosine monophosphate, creatine, betaine, carnosine and hypoxanthine) out of eighteen metabolites differ significantly (p < 0.05) in content depending on the aging type. Fifteen metabolites in dry-aged and ten in wet-aged beef correlate with the aging time (r > 0.7, <-0.7), which shows significant aging time-related effects on the polar fraction of metabolome.

The dry aged beef paradox: Why dry aging is sometimes not better than wet aging.

An increasing consumer demand for a higher quality and eating experience has led to a revisit to the dry aging process. Therefore, research also focuses on the effects of different dry aging methods and aims to improve the dry aging process. However, an optimal process cannot be defined and, unfortunately, most of the dry aging results only hold true for the individual experiment. If one repeats a dry aging process in a different facility, the result might differ. The same dry aging process in two different ripening chambers does not inevitably contribute to equal tenderness and flavor. Since this is still not been understood well, this review presents results of the most relevant dry aging studies by illustrating different process parameters and cuts. Some conclusions which may be useful to explain the paradox are derived from the literature in order to understand the crucial factors and commonalities in the dry aging process.

A human ESC-based screen identifies a role for the translated lncRNA LINC00261 in pancreatic endocrine differentiation.

Long noncoding RNAs (lncRNAs) are a heterogenous group of RNAs, which can encode small proteins. The extent to which developmentally regulated lncRNAs are translated and whether the produced microproteins are relevant for human development is unknown. Using a human embryonic stem cell (hESC)-based pancreatic differentiation system, we show that many lncRNAs in direct vicinity of lineage-determining transcription factors (TFs) are dynamically regulated, predominantly cytosolic, and highly translated. We genetically ablated ten such lncRNAs, most of them translated, and found that nine are dispensable for pancreatic endocrine cell development. However, deletion of LINC00261 diminishes insulin+ cells, in a manner independent of the nearby TF FOXA2. One-by-one disruption of each of LINC00261's open reading frames suggests that the RNA, rather than the produced microproteins, is required for endocrine development. Our work highlights extensive translation of lncRNAs during hESC pancreatic differentiation and provides a blueprint for dissection of their coding and noncoding roles.

High-pressure processing of usually discarded dry aged beef trimmings for subsequent processing.

In this study, we investigated if the usually discarded trimmings from dry aged beef can be incorporated into raw fermented sausages as a substitute for fresh beef without altering any major characteristics. Dry aged trimmings were subjected to high-pressure processing (600 MPa, 3 min hold) to reduce the bacterial load, achieving a 3-log reduction. HPP-treated dry aged beef trimmings were then incorporated into raw fermented sausages (60% pork and 40% beef). Beef was substituted with trimmings in different concentrations (7.5, 12.5, 25, 50, 100%). Due to the substitution, the water content of the sausages was reduced depending on the amount of beef substituted. Consequently, the sausages with substituted beef, for example, 50 and 100%, achieved the same water content after 5.4 and 3.7 days, respectively, than control sausage at day 9. However, the substitution (100%) affected the fat content, which contributes to significant differences (p < .05) in firmness during ripening.

Thyrotropin Receptor: Allosteric Modulators Illuminate Intramolecular Signaling Mechanisms at the Interface of Ecto- and Transmembrane Domain.

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.

Widespread Translational Control of Fibrosis in the Human Heart by RNA-Binding Proteins.

BACKGROUND: 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. METHODS: 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. RESULTS: We generated nucleotide-resolution translatome data during the transforming growth factor ß1-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 ß1. CONCLUSIONS: We reveal widespread translational effects of transforming growth factor ß1 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.

The Translational Landscape of the Human Heart.

Gene expression in human tissue has primarily been studied on the transcriptional level, largely neglecting translational regulation. Here, we analyze the translatomes of 80 human hearts to identify new translation events and quantify the effect of translational regulation. We show extensive translational control of cardiac gene expression, which is orchestrated in a process-specific manner. Translation downstream of predicted disease-causing protein-truncating variants appears to be frequent, suggesting inefficient translation termination. We identify hundreds of previously undetected microproteins, expressed from lncRNAs and circRNAs, for which we validate the protein products in vivo. The translation of microproteins is not restricted to the heart and prominent in the translatomes of human kidney and liver. We associate these microproteins with diverse cellular processes and compartments and find that many locate to the mitochondria. Importantly, dozens of microproteins are translated from lncRNAs with well-characterized noncoding functions, indicating previously unrecognized biology.