Dynamic protein composition of Saccharomyces cerevisiae ribosomes in response to multiple stress conditions reflects alterations in translation activity.

Ribosomes, intercellular macromolecules responsible for translation in the cell, are composed of RNAs and proteins. While rRNA makes the scaffold of the ribosome and directs the catalytic steps of protein synthesis, ribosomal proteins play a role in the assembly of the subunits and are essential for the proper structure and function of the ribosome. To date researchers identified heterogeneous ribosomes in different developmental and growth stages. We hypothesized that under stress conditions the heterogeneity of the ribosomes may provide means to prepare the cells for quick recovery. Therefore the aim of the study was the identification of heterogeneity of ribosomal proteins within the ribosomes in response to eleven stress conditions in Saccharomyces cerevisiae, by means of a liquid chromatography/high resolution mass spectrometry (LC-HRMS) and translation activity tests. Out of the total of 74 distinct ribosomal proteins identified in the study 14 small ribosomal subunit (RPS) and 8 large ribosomal subunit (RPL) proteins displayed statistically significant differential abundances within the ribosomes under stress. Additionally, significant alterations in the ratios of 7 ribosomal paralog proteins were observed. Accordingly, the translational activity of yeast ribosomes was altered after UV exposure, during sugar starvation, cold shock, high salt, anaerobic conditions, and amino acid starvation.

Exploring dog antler chews as a novel source of collagen supplementation: extraction and characterization of collagen from red deer antlers.

Collagen is the body's most abundant protein and is primarily found in the skin, bones, tendons, and ligaments of animals and fish. As the interest in collagen supplementation grows, new sources of this protein are continually being introduced. We have confirmed that red deer antlers are a source of type I collagen. We investigated the effects of chemical treatment, temperature, and time on the extractability of collagen from red deer antlers. The optimal conditions for obtaining the highest collagen yield were determined to be: 1) removing noncollagenous proteins at 25°C for 12 h in an alkaline solution, 2) defatting at 25°C using a 1:10 grounded antler:butyl alcohol ratio, and 3) acidic extraction lasting 36 h using a 1:10 antler:acetic acid ratio. Under these conditions, we obtained a collagen yield of 22.04%. The molecular characterization of red deer antler collagen revealed typical features of type I collagens, including the presence of three α-chains, high glycine content, and high levels of proline and hydroxyproline, as well as helical arrangements. This report suggests that red deer antlers have significant potential as a source of collagen supplements.

The Evaluation of SHAPE-MaP RNA Structure Probing Protocols Reveals a Novel Role of Mn2+ in the Detection of 2'-OH Adducts.

Chemical probing, for decades, has been one of the most popular tools for studying the secondary structure of RNA molecules. Recently, protocols for simultaneous analysis of multiple RNAs have been developed, enabling in vivo transcriptome-wide interrogation of the RNA structure dynamics. One of the most popular methods is the selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP). In this study, we describe the evaluation of this protocol by addressing the influence of the reverse transcription enzymes, buffer conditions, and chemical probes on the properties of the cDNA library and the quality of mutational profiling-derived structural signals. Our results reveal a SuperScript IV (SSIV) reverse transcriptase as a more efficient enzyme for mutational profiling of SHAPE adducts and shed new light on the role of Mn2+ cations in the modulation of SSIV readthrough efficiency.

The emerging roles of tRNAs and tRNA-derived fragments during aging: Lessons from studies on model organisms.

Aging is a gradual decline of various functions of organisms resulting in diminished abilities to protect against the environmental damage and reinforce the physiological harmony. Age-related functional declines have been thought to be passive and not regulated. However, studies on numerous model organisms, from yeast to mammals, exposed that the mechanisms of lifespan regulation are remarkably conserved throughout the evolution. Following the pioneering genetic studies in C. elegans, it has been shown that the genes related to the longevity are conserved in yeast, flies and mice. For a long time, tRNAs have been only considered as molecules transporting amino acids to the ribosome during translation. Nonetheless, it has become apparent from many biological studies that tRNAs are entangled in a variety of physiological and pathological processes. This review focuses on the emerging roles of tRNA-associated processes in aging and lifespan of model organisms. More specificaly, we present a summary on the importance of tRNA metabolism, epitranscriptome and possible roles of tRNA-derived fragments in aging and lifespan regulation. Better understanding of the basic mechanisms of aging could lead to the development of new diagnostics and treatments for aging-related diseases.

tRNA-derived fragments as new players in regulatory processes in yeast.

For a very long time, RNA molecules were treated as transistory molecules, by which the genetic information flows from DNA to proteins; the model proposed in the 1960s accepted that proteins are both the products and the regulators of gene expression. Since then, thousands of reports proved that RNAs should be thought about as the factors that do control gene expression. The pervasive transcription has been reported in many eukaryotic organisms, illustrating a highly interwoven transcriptome organization that includes hundreds of previously unknown noncoding RNAs. The key roles of noncoding RNAs (microRNAs and small interfering RNAs) in gene expression regulation are no longer surprising, as are new classes of noncoding RNAs constantly being discovered. Transfer RNAs (tRNAs) are the second most abundant type of RNAs in the cell. Advances in high-throughput sequencing technologies exposed the existence of functional, regulatory tRNA-derived RNA fragments (tRFs), generated from precursor and mature tRNAs. These tRF molecules have been found to play central roles during stress and different pathological conditions. Herein, we present the critical assessment of the discoveries made in the field of tRNA-derived fragments in the past 15 years in various pathogenic and nonpathogenic yeast species.

Autologous Matrix-Induced Chondrogenesis (AMIC) for Focal Chondral Lesions of the Knee: A 2-Year Follow-Up of Clinical, Proprioceptive, and Isokinetic Evaluation.

(1) Background: The autologous matrix-induced chondrogenesis (AMIC) is a bio-orthopedic treatment for articular cartilage damage. It combines microfracture surgery with the application of a collagen membrane. The aim of the present study was to report a medium-term follow-up of patients treated with AMIC for focal chondral lesions. (2) Methods: Fourty-eight patients treated surgically and 21 control participants were enrolled in the study. To evaluate the functional outcomes, the proprioceptive (postural stability, postural priority) and isokinetic (peak value of maximum knee extensor and flexor torque in relation to body mass and the total work) measurements were performed. To evaluate the clinical outcomes, the Lysholm score and the IKDC score were imposed. (3) Results: Compared to the preoperative values, there was significant improvement in the first 2 years after intervention in the functional as well as subjective outcome measures. (4) Conclusions: AMIC showed durable results in aligned knees.

Intra-Articular Injections of Autologous Adipose Tissue or Platelet-Rich Plasma Comparably Improve Clinical and Functional Outcomes in Patients with Knee Osteoarthritis.

The use of biologic therapies for the management of knee osteoarthritis (OA) has largely increased in recent years. The purpose of this study was to evaluate the efficiency and the therapeutic potential of platelet-rich plasma (PRP) and autologous adipose tissue (AAT) injections as a treatment for knee OA. Sixty participants were enrolled in the study: 20 healthy ones and 40 with minimal to moderate knee OA (KL I-III). The OA patients were randomly assigned either to the PRP or to the AAT group. The PRP samples showed a low expression level of NF-κB-responsive gene CCL5 and high expression levels of classic inflammatory and TNF-l INF responses. The AAT injection product was prepared using a Lipogems device, and its regenerative potential as well as the ability for expansion of mesenchymal stem cells were tested in the cell culture conditions. The patient assessments were carried out five times. Significant improvement was observed regardless of the treatment method in the VAS, KOOS, WOMAC and IKDC 2000 subjective evaluations as well as in the functional parameters. Intra-articular injections of AAT or PRP improved pain, symptoms, quality of life and functional capacity with a comparable effectiveness in the patients with mild to moderate knee osteoarthritis.

PTT-quant: a new method for direct identification and absolute quantification of premature transcription termination events, following the example of bacterial riboswitches.

Regulation of gene expression by premature termination of transcription has been well described in all domains of life, including metazoans, yeast, plants, and bacteria. Although methods for identification of such regulatory events by sequencing are available, the focused biochemical studies of the mechanism are hampered by lack of highly sensitive and accurate experimental methods. Here, we propose a new method for absolute quantification of premature transcription termination events, PTT-quant. It is based on highly sensitive two-step digital droplet PCR protocol, coupled with normalized cDNA synthesis attained by site-specific pre-cleavage of investigated transcripts with RNase H. As a consequence, our method enables the reliable and sensitive quantification of both, prematurely terminated and full-length transcripts. By application of our method to investigation of transcriptional riboswitches in Bacillus subtilis, we were able to precisely measure the dynamics of S-adenosylmethionine (SAM) riboswitch induction, which turned to be ~ 23% higher in comparison the results obtained without cDNA synthesis normalization.Key points• A novel method for quantification of premature transcription termination events was established.• PTT-quant measures absolute concentration of full-length and terminated transcripts.• RNase H and the digital droplet PCR technique is used in PTT-quant.

Ribosome heterogeneity as a new element of translation regulation / Heterogenicznosc rybosomów jako nowy element regulacji translacji.

All living cells depend on the fine-tuning of gene expression and protein biosynthesis. Ribosomes, the molecular machines at the center of translation, have been previously considered the invariable driving force of protein production. However, recent studies indicated that the ribosomes are actively involved in the regulation of translation, influencing the control of translation initiation, the elongation speed, and the mRNA translation selectivity. This is due to the presence of subpopulations of the ribosomes, which differ in rRNAs and protein composition, their modifications and protein stoichiometry. In this publication, we focused our attention on the ribosomal heterogeneity in eukaryotes, which results from the changes in the stoichiometry of the ribosomal proteins and the existence of protein paralogs.