Ghrelin Immunoreactive Cell Amounts in the Abomasum in 4-Month-Old Calves by Feeding Different Amounts of Prebiotics and New Synbiotics

The study aim was to determine prebiotic (inulin) and new synbiotic (inulin and Enterococcus faecium) varied dosage effects, during food breakdown-abomasum immunoreactive (IR) cell amount and cold carcass weight. Ghrelin is synthesized in the fundus region of the stomach. In the gastrointestinal system, ghrelin affects multiple functions, including secretion of gastric acid, gastric motility, and pancreatic protein output. The study consisted of 49 Holstein male calves (23 ± 5 days old, 50 ± 5 kg). Control and experimental groups were differentiated only with the additive amount added to the morning food source. Three prebiotic groups were fed Jerusalem artichoke flour (inulin content increased by 50%) in three amounts: 6 g (lowest) PreG6, 12 g (medium) PreG12, and 24 g (highest) PreG24. Three synbiotic groups were added 0.25 g of prebiotic Enterococcus faecium (2 ∗ 109 CFU/g) to the respective prebiotic, obtaining a new synbiotic (SynG6, SynG12, and SynG24). Calves were slaughtered after 56 days to obtain abomasum samples for ghrelin IR cell examination, and carcass weight was determined. It shows that ghrelin IR cell count in the abomasum was ( p < 0.05 ) reduced in 6g and 12g inulin dosage, but carcass weight was significantly ( p < 0.05 ) higher for PreG12 and PreG24 ( p < 0.05 ) and then for CoG (CoG 42.6 kg; PreG12 51.4 kg; and PreG24 54.0 kg) and ( p < 0.05 ) for SynG12 and SynG24 (SynG12 52.3 kg and SynG24 49.6 kg), which indicates longer satiety and more wholesome breakdown of the food uptake. It was concluded that ghrelin IR cells in 12-week-old calves are more abundant in the fundus region. Medium- and high-dosage prebiotic inulin feeding to the calves improves overall food digestion, allowing for longer satiety and higher cold carcass weight without increasing food amount. Adding synbiotic 0.25 g Enterococcus faecium (2 ∗ 109 CFU/g (Protexin, UK)) to inulin (produced in Latvia LTD „Herbe”) does not improve the results of this prebiotic.

Wobble tRNA modification and hydrophilic amino acid patterns dictate protein fate

Regulation of mRNA translation elongation impacts nascent protein synthesis and integrity and plays a critical role in disease establishment. Here, we investigate features linking regulation of codon-dependent translation elongation to protein expression and homeostasis. Using knockdown models of enzymes that catalyze the mcm5s2 wobble uridine tRNA modification (U34-enzymes), we show that gene codon content is necessary but not sufficient to predict protein fate. While translation defects upon perturbation of U34-enzymes are strictly dependent on codon content, the consequences on protein output are determined by other features. Specific hydrophilic motifs cause protein aggregation and degradation upon codon-dependent translation elongation defects. Accordingly, the combination of codon content and the presence of hydrophilic motifs define the proteome whose maintenance relies on U34-tRNA modification. Together, these results uncover the mechanism linking wobble tRNA modification to mRNA translation and aggregation to maintain proteome homeostasis.

Combinatorial optimization of mRNA structure, stability, and translation for RNA-based therapeutics

SUMMARYTherapeutic mRNAs and vaccines are being developed for a broad range of human diseases, including COVID-19. However, their optimization is hindered by mRNA instability and inefficient protein expression. Here, we describe design principles that overcome these barriers. We develop a new RNA sequencing-based platform called PERSIST-seq to systematically delineate in-cell mRNA stability, ribosome load, as well as in-solution stability of a library of diverse mRNAs. We find that, surprisingly, in-cell stability is a greater driver of protein output than high ribosome load. We further introduce a method called In-line-seq, applied to thousands of diverse RNAs, that reveals sequence and structure-based rules for mitigating hydrolytic degradation. Our findings show that “superfolder” mRNAs can be designed to improve both stability and expression that are further enhanced through pseudouridine nucleoside modification. Together, our study demonstrates simultaneous improvement of mRNA stability and protein expression and provides a computational-experimental platform for the enhancement of mRNA medicines.

Lubricating properties of chewing stimulated whole saliva from patients suffering from xerostomia

Objectives The study aimed to quantify the lubricating properties of chewing stimulated whole saliva from healthy controls (n = 22), from patients suffering from primary Sjögren’s syndrome (n = 37) and from patients undergoing head-and-neck radiotherapy (n = 34). Materials and methods All participants had to complete the Xerostomia Inventory questionnaire to score dry mouth sensation. Lubrication was measured using an ex vivo tongue-enamel friction system in terms of Relief and Relief period. MUC5b and total protein concentrations of the saliva samples were measured by an enzyme-linked immunosorbent assay and a bicinchoninic acid assay, respectively. Results Relief of Sjögren’s patients’ saliva and post-irradiation patients’ saliva was similar compared with healthy controls, but saliva from post-irradiation patients lubricated significantly better than saliva from Sjögren’s patients. The Relief period was similar between the three groups. The Relief and Relief period were higher for saliva samples post-irradiation compared to pre-irradiation. MUC5b and total protein concentrations were comparable in all groups. MUC5b and total protein output were significantly lower in patients subjected to radiotherapy compared to saliva from healthy controls and pre-irradiation patients. MUC5b concentrations positively correlated with lubricating properties of post-irradiation patient saliva. Conclusions The lubricating properties of patient saliva were not any worse than healthy controls. Lower flow rate leads to lower availability of saliva in the oral cavity and decreases the overall output of protein and MUC5b, which might result in an insufficient replenishing of the mucosal salivary film. Clinical relevance An insufficient replenishing might underlie the sensation of a dry mouth and loss of oral function.

Biotribological properties of xerostomia patient saliva and its enhancement

The study aimed to quantify the lubricating properties of chewing stimulated whole saliva from healthy controls (n=22), from patients suffering from primary Sjögren’s syndrome (n=37) and from patients undergoing head-and-neck radiotherapy (n=34). Materials and Methods All participants had to complete the Xerostomia Inventory questionnaire to score dry mouth sensation. Lubrication was measured using an ex vivo tongue-enamel friction system in terms of Relief and Relief period. MUC5b and total protein concentrations of the saliva samples were measured by an enzyme-linked immunosorbent assay and a bicinchoninic acid assay, respectively. Results Relief of Sjögren’s patients saliva and post-irradiation patients saliva was similar compared with healthy controls, but saliva from post-irradiation patients lubricated significantly better than saliva from Sjögren’s patients. The Relief period was similar between the three groups. The Relief and Relief period were higher for saliva samples post-irradiation compared to pre-irradiation. MUC5b and total protein concentrations were comparable in all groups. MUC5b and total protein output were significantly lower in patients subjected to radiotherapy compared to saliva from healthy controls and pre-irradiation patients. MUC5b concentrations positively correlated with lubricating properties of post-irradiation patient saliva. Conclusions The lubricating properties of patient saliva were not any worse than healthy controls. Lower flow rate leads to lower availability of saliva in the oral cavity and decreases the overall output of protein and MUC5b, which might result in an insufficient replenishing of the mucosal salivary film. Clinical Relevance An insufficient replenishing might underlie the sensation of a dry mouth and loss of oral function. In the talk I will explain biomaterials related strategies, yet ex vivo, to enhance salivary lubrication despite of low flowrates.

A translational program that suppresses metabolism to shield the genome

Translatome reprogramming is a primary determinant of protein levels during stimuli adaptation. This raises the question: what are the translatome remodelers that reprogram protein output to activate biochemical adaptations. Here, we identify a translational pathway that represses metabolism to safeguard genome integrity. A system-wide MATRIX survey identified the ancient eIF5A as a pH-regulated translation factor that responds to fermentation-induced acidosis. TMT-pulse-SILAC analysis identified several pH-dependent proteins, including the mTORC1 suppressor Tsc2 and the longevity regulator Sirt1. Sirt1 operates as a pH-sensor that deacetylates nuclear eIF5A during anaerobiosis, enabling the cytoplasmic export of eIF5A/Tsc2 mRNA complexes for translational engagement. Tsc2 induction inhibits mTORC1 to suppress cellular metabolism and prevent acidosis-induced DNA damage. Depletion of eIF5A or Tsc2 leads to metabolic re-initiation and proliferation, but at the expense of incurring substantial DNA damage. We suggest that eIF5A operates as a translatome remodeler that suppresses metabolism to shield the genome.

Crystal Structure of a Variant PAM2 Motif of LARP4B Bound to the MLLE Domain of PABPC1

Eukaryotic cells determine the protein output of their genetic program by regulating mRNA transcription, localization, translation and turnover rates. This regulation is accomplished by an ensemble of RNA-binding proteins (RBPs) that bind to any given mRNA, thus forming mRNPs. Poly(A) binding proteins (PABPs) are prominent members of virtually all mRNPs that possess poly(A) tails. They serve as multifunctional scaffolds, allowing the recruitment of diverse factors containing a poly(A)-interacting motif (PAM) into mRNPs. We present the crystal structure of the variant PAM motif (termed PAM2w) in the N-terminal part of the positive translation factor LARP4B, which binds to the MLLE domain of the poly(A) binding protein C1 cytoplasmic 1 (PABPC1). The structural analysis, along with mutational studies in vitro and in vivo, uncovered a new mode of interaction between PAM2 motifs and MLLE domains.

THE GROWTH RETARDATION EFFECT IN EARLY HEIFERS ONTOGENESIS ON DAIRY COWS PRODUCTIVITY

The objective of the study is to determine, whether the growth retardations of replacement cows are admissible, and in which periods of culture they most significantly affect the further productivity of the cows. The subject of the analysis was the number of animals who at the age of 15 months had the live weight no less than 300 kg (which complied with the breed standard or was inferior to the requirements for 10 % maximum). The animals were divided into 5 groups. The first group included the animals with growth retardation before the age of 3 months. The second one – at the age of 3-6 months; the third one – 6-9 months and the fourth one – 9-12 months. The fifth one was the control group, without growth retardation. The growth retardation referred to the live weight reduction at the end of the period in comparison with its beginning or the average daily weight gain less than 500 g. The authors measured the live weight, first lactation productivity and the lifetime productivity of the animals. It has been established that the growth retardation of the heifers before the age of 3 months negatively affected the growth rate in the following three-month period. They can completely compensate the growth retardation only at the age of 18 months. The other group animals compensated the live weight retardation in comparison with the control group by the age of 15 months. The firstlings which had the growth retardation at the age of 0-3 and 3-6 months, has the highest milk productivity. The same trend was observed in milk fat and protein output and the age of the first calving. However, in the growth retardation group the livability of firstlings, the number of lactations and the productive live was worse. The lifelong milk yield in the growth retardation group was 15-37 % lower than in the control group. Growth retardation at the age of 6-9 months negatively affected the higher lactation productivity. The highest daily milk yield in this group of cows was lower in the control group by 14 % (p < 0.05). Therefore, growth retardation had no negative effect on the milk yield of the firstlings, but resulted in lower survival of the animals, shortened the productive life and lifetime yield. The later the growths retardation occurs, the sooner the heifers can compensate it and the lower is its effect on the lifetime productivity of the cows. In view of the reduction of lifetime productivity, it is purposeful to draft out the animals with the growth retardation.

Limited but gene-class specific correlation of mRNA and protein expression in human CD8+ T cells

SUMMARYT cell differentiation and activation induces substantial alterations in gene expression. While RNA sequencing and single cell RNA sequencing analysis provided important insights in the gene expression dynamics of T cells, it is not well understood how the mRNA expression translates into the protein landscape. By combining paired RNA-sequencing and mass spectrometry data of primary human CD8+ T cells, we found that mRNA expression is a poor proxy for the overall protein output. Irrespective of the differentiation or activation status, the correlation coefficient of human CD8+ T cells reached a mere 0.41-0.43. Only gene classes that mediate conserved cellular processes such as protein translation or cellular metabolism showed a strong correlation of mRNA with protein expression. In contrast, the mRNA expression and protein output of transcription factors, cell surface molecules, and secreted proteins - including cytokines - only mildly correlated. Conversely, highly conserved genes correlated well with the protein output. This was also true for the presence of AU-rich elements in the 3’untranslated region, in particular for mRNAs that encode secreted proteins. In conclusion, the in-depth characterization of the transcriptome and proteome in human CD8+ T cells emphasizes the need of combined mRNA and protein analysis for our understanding of T cell biology and function.