Biological strategies supporting the orthopaedic treatment / Biologiczne wspomaganie leczenia ortopedycznego.

Damage to the musculoskeletal system significantly impairs mobility and quality of life, limiting everyday activities. For a successful orthopedic treatment, anatomical, physiological and biomechanical factors must be taken into account as they all influence tissue healing. It is therefore of crucial importance to support traditional treatment with biological therapies, as they facilitate the regeneration of the tissue microarchitecture. Such orthobiologics work at the cellular level (orthobiologics rich in mesenchymal cells or growth factors) or at the tissue level (matrices for repairing e.g. cartilage). In this review, we describe the most frequently used orthobiologics rich in mesenchymal cells (bone marrow, autologous adipose tissue, tenocytes, umbilical cord, urine and bursa) and growth factors, presenting the molecular basis of their functioning and their clinical effectiveness.

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.

Genetics of antibiotic resistance in methicillin-resistant Staphylococcus aureus (MRSA).

Methicillin-resistant Staphylococcus aureus (MRSA) strains pose a significant threat as common causes of bacterial infections in hospitals, often resistant to available antibiotics such as daptomycin, vancomycin, and linezolid. The continuous emergence of new MRSA isolates with no effective treatment options underscores a real threat to health among humans and animals, and the number of effective antibiotic therapies decreases with each passing year. In this review, we provide an overview of the most common genetic mechanisms of resistance to a broad spectrum of antibiotics in methicillin-resistant S. aureus.

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.

Agricultural biotechnology for sustainable food security.

Of late, global food security has been under threat by the coronavirus disease 2019 (COVID-19) pandemic and the recent military conflict in Eastern Europe. This article presents the objectives of the Sustainable Development Goals and the European Green Deal related to achieving food security and sustainable development in European Union (EU) agriculture, taking the aforementioned threats into account. In addition, it discusses the future of plant agricultural biotechnology and artificial intelligence (AI) systems, considering their potential for reaching the goal of food security. Paradoxically, the present challenging situation may allow politicians and stakeholders of the EU to realize opportunities and use the potential of the biotechnology sector.

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.

Atypical DNA methylation of genes encoding cysteine-rich peptides in Arabidopsis thaliana.

BACKGROUND: In plants, transposons and non-protein-coding repeats are epigenetically silenced by CG and non-CG methylation. This pattern of methylation is mediated in part by small RNAs and two specialized RNA polymerases, termed Pol IV and Pol V, in a process called RNA-directed DNA methylation. By contrast, many protein-coding genes transcribed by Pol II contain in their gene bodies exclusively CG methylation that is independent of small RNAs and Pol IV/Pol V activities. It is unclear how the different methylation machineries distinguish between transposons and genes. Here we report on a group of atypical genes that display in their coding region a transposon-like methylation pattern, which is associated with gene silencing in sporophytic tissues. RESULTS: We performed a methylation-sensitive amplification polymorphism analysis to search for targets of RNA-directed DNA methylation in Arabidopsis thaliana and identified several members of a gene family encoding cysteine-rich peptides (CRPs). In leaves, the CRP genes are silent and their coding regions contain dense, transposon-like methylation in CG, CHG and CHH contexts, which depends partly on the Pol IV/Pol V pathway and small RNAs. Methylation in the coding region is reduced, however, in the synergid cells of the female gametophyte, where the CRP genes are specifically expressed. Further demonstrating that expressed CRP genes lack gene body methylation, a CRP4-GFP fusion gene under the control of the constitutive 35 S promoter remains unmethylated in leaves and is transcribed to produce a translatable mRNA. By contrast, a CRP4-GFP fusion gene under the control of a CRP4 promoter fragment acquires CG and non-CG methylation in the CRP coding region in leaves similar to the silent endogenous CRP4 gene. CONCLUSIONS: Unlike CG methylation in gene bodies, which does not dramatically affect Pol II transcription, combined CG and non-CG methylation in CRP coding regions is likely to contribute to gene silencing in leaves because loss of this methylation in synergid cells is associated with CRP gene expression. We discuss this unusual methylation pattern and its alteration in synergid cells as well as the possible retrogene origin and evolutionary significance of CRP genes that are methylated like transposons.

Public opinion on biotechnology and genetic engineering in the European Union: Polish consumer study.

The aim of the article is to show the current state of public opinion of Poles on biotechnology and genetic engineering in the context of European Union countries. The authors refer to the results of their own study based on a survey conducted in 2019 in Poland. To introduce the matter of public opinion on biotechnology and genetic engineering in the European Union a short review of research related to the topic is presented, showing discrepancies in perception of biotechnology and genetic engineering. The results of the survey showed that more than half of Poles noticed that products obtained by genetic engineering techniques are available on the market. Despite the fear of the research in the field of biotechnology and genetic engineering, 39 to 69% (depending on the subject of research) of Poles supported them. Moreover, 62% of Poles were opponents of genetically modified feeds as they believed that they can be harmful to human life and health. The findings regarding the current consumer perception, knowledge, and attitude towards genetically modified foods and feeds will help in building strategic approaches to educating society about genetically modified organisms and genetically modified products.

Bioeconomy development factors in the European Union and Poland.

Bioeconomy is not an autonomous sector of the economy, but rather a complex mechanism involving agriculture, industry, biotechnology, service sectors and consumers. To measure the size of the bioeconomy in European Union (EU) countries, it is necessary to create appropriate indicators that allow it to be monitored with reference to its current state, growth rate and sector description. In many countries, including Poland, there is no complete information or data collection system to monitor bioeconomy development directly, e.g. in the Polish Central Statistical Office. In response to these needs, several groups of indicators related to the circular economy, sustainable development and Europe 2020 were created by the European Commission (EC) in the Eurostat database. These indicators can help monitoring of bioeconomy development in EU countries. The present study discusses factors for bioeconomy development through an analysis of their social, economic and environmental aspects, as well as showing the value of the selected indicators in the EU and Poland. In addition, a separate section is dedicated to public perception of bioeconomy and to legislation regarding genetically modified organisms (GMOs). To date, many research studies have been reported on the public acceptance of bioeconomy issues in the EU, including renewable resources, biofuels, GMOs, bio-based products, food security and climate change. The awareness and perception of society on the bioeconomy, bio-based products and processes, and the sustainable use of resources can contribute to environmental sustainability, but intensified efforts are required to increase public acceptance.

Herbicide stress-induced DNA methylation changes in two Zea mays inbred lines differing in Roundup® resistance.

DNA methylation plays a crucial role in the regulation of gene expression, activity of transposable elements, defense against foreign DNA, and inheritance of specific gene expression patterns. The link between stress exposure and sequence-specific changes in DNA methylation was hypothetical until it was shown that stresses can induce changes in the gene expression through hypomethylation or hypermethylation of DNA. To detect changes in DNA methylation under herbicide stress in two local Zea mays inbred lines exhibiting differential susceptibility to Roundup®, the methylation-sensitive amplified polymorphism (MSAP) technique was used. The overall DNA methylation levels were determined at approximately 60% for both tested lines. The most significant changes were observed for the more sensitive Z. mays line, where 6 h after the herbicide application, a large increase in the level of DNA methylation (attributed to the increase in fully methylated bands (18.65%)) was noted. DNA sequencing revealed that changes in DNA methylation profiles occurred in genes encoding heat shock proteins, membrane proteins, transporters, kinases, lipases, methyltransferases, zinc-finger proteins, cytochromes, and transposons. Herbicide stress-induced changes depended on the Z. mays variety, and the large increase in DNA methylation level in the sensitive line resulted in a lower ability to cope with stress conditions.

Identification of Structural Variants in Two Novel Genomes of Maize Inbred Lines Possibly Related to Glyphosate Tolerance.

To study genetic variations between genomes of plants that are naturally tolerant and sensitive to glyphosate, we used two Zea mays L. lines traditionally bred in Poland. To overcome the complexity of the maize genome, two sequencing technologies were employed: Illumina and Single Molecule Real-Time (SMRT) PacBio. Eleven thousand structural variants, 4 million SNPs and approximately 800 thousand indels differentiating the two genomes were identified. Detailed analyses allowed to identify 20 variations within the EPSPS gene, but all of them were predicted to have moderate or unknown effects on gene expression. Other genes of the shikimate pathway encoding bifunctional 3-dehydroquinate dehydratase/shikimate dehydrogenase and chorismate synthase were altered by variants predicted to have a high impact on gene expression. Additionally, high-impact variants located within the genes involved in the active transport of glyphosate through the cell membrane encoding phosphate transporters as well as multidrug and toxic compound extrusion have been identified.

Analysis of miRNA expression associated with the Lr46 gene responsible for APR resistance in wheat (Triticum aestivum L.).

Lr46/Yr29/Pm39 (Lr46) is a gene for slow rusting resistance in wheat. The aim of the study was to analyze the miRNA expression in selected common wheat cultivars carrying resistance genes, Lr46 among others (HN Rod, Pavon'S', Myna'S', Frontana'S', and Sparrow'S') in response to leaf rust infection caused by Puccinia triticina Erikss. In the Pavon 'S', Myna 'S', Frontana'S', and Sparow'S' varieties a product with a length of 242 bp has been identified, which is specific to the Xwmc44 marker linked to the brown rust resistance gene Lr46. In the next step, the differences in the expression of microRNA (miR5085 and miR164) associated with the Lr46 gene, which is responsible for different resistance of selected wheat cultivars to leaf rust, were examined using emulsion PCR (ddPCR). In the experiment, biotic stress was induced in mature plants by infecting them with fungal spores under controlled conditions in a growth chamber. For analysis the plant material was collected before inoculation and 6, 12, 24, and 48 h after inoculation. The experiments also showed that plant infection with Puccinia triticina resulted in an increase in miR164 expression in cultivars carrying the Lr46 gene. The expression of miR164 remained stable in a control cultivar (HN ROD) lacking this gene. This has proved that miR164 can be involved in leaf rust resistance mechanisms.

Certain new plant breeding techniques and their marketability in the context of EU GMO legislation - recent developments.

The comparatively low adoption rate of GMO products in the European Union (EU) market seems to be connected with the strictness of authorization regulations and inefficiency of the authorization process itself. These problems will apply to any product deemed to be a GMO that could potentially be marketable in the EU. Since modern methods of plant breeding involving oligonucleotide-directed mutagenesis (ODMs) or site-directed nucleases (SDNs), including Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), are becoming ever more popular, it is crucial to establish whether the products of such new breeding techniques (NBTs), in particular those which involve precise methods of mutagenesis, are exempted from the EU legislation on GMOs or not. Legal uncertainty as to their status may result in reluctance to invest in such methods and develop them further. Here, developments are presented in the legal classification of certain NBTs products in the context of recent decisions and jurisprudence. The socioeconomic aspects of GMO adoption in both global and European contexts are discussed. The legal and practical landscape of GMO regulation in the EU is presented and how it may pose an obstacle to investment and the development of new products. The latest jurisprudence (e.g., Case C-528/16) [1] on the interpretation of the legal concept of GMOs and the scope of the legislation are analyzed, with the conclusion that the strict regulations will probably also apply to products of the NBTs involving precise methods of mutagenesis. This in turn will probably result in the restriction of their application in the development of new plant varieties in the EU.

Towards Food Security: Current State and Future Prospects of Agrobiotechnology.

The consistent increase in the global population, estimated to reach 9 billion people by 2050, poses a serious challenge for the achievement of global food security. Therefore, the need to feed an increasing world population and to respond adequately to the effects of climate change must be urgently considered. Progress may be achieved by applying knowledge of molecular and genetic mechanisms to create and/or improve agricultural and industrial processes. We highlight the importance of crops (wheat, maize, rice, rapeseed, and soybean) to the development of sustainable agriculture and agrobiotechnology in the EU and discuss possible solutions for ensuring food security, while also considering their social acceptance.

[Mechanism of peptide bond formation on the ribosome--controversions]. / Kontrowersje wokól mechanizmu syntezy wiazania peptydowego w rybosomie.

During past five years there have been published many experimental data concerning structure and function of the ribosome. With the presentation of atomic structures we obtained a new data about composition of peptidyl transferase center. It is now obvious that PTC is composed entirely of rRNA. It is also known that the proper substrate alignment is the major factor for ribosome's catalytic activity. However, more detailed mechanism of peptide bond formation on the ribosome still remains unclear. Several issues remain unsolved. For example, are there any chemical components coming from ribosome itself, that enhance the rate of the reaction? Do intact ribosomes perform peptidyltransfer in the same way as the isolated ribosomal subunits that have been the source of most of the data? In this article we present different opinions and controversions around peptide bond formation on the ribosome.

[The influence of small regulatory RNAs on the viral infection--new strategies in HCV therapy]. / Wplyw malych regulatorowych RNA na przebieg zakazen wirusowych--nowe strategie leczenia zakazen HCV.

The discovery of the RNAi phenomenon drastically changed our thinking about the mechanisms affecting the expression of genetic information. It demonstrated that not only proteins but also small regulatory RNAs (srRNAs) are the key players in this process. In addition, it was shown that srRNAs can influence viral infections. They are able to mediate viral RNA degradation or to inhibit viral RNA replication and translation. The collected data suggest that srRNAs will soon become an important tool in our struggle with viruses.

Deciphering the soybean molecular stress response via high-throughput approaches.

As a result of thousands of years of agriculture, humans had created many crop varieties that became the basis of our daily diet, animal feed and also carry industrial application. Soybean is one of the most important crops worldwide and because of its high economic value the demand for soybean products is constantly growing. In Europe, due to unfavorable climate conditions, soybean cultivation is restricted and we are forced to rely on imported plant material. The development of agriculture requires continuous improvements in quality and yield of crop varieties under changing or adverse conditions, namely stresses. To achieve this goal we need to recognize and understand the molecular dependencies underlying plant stress responses. With the advent of new technologies in studies of plant transcriptomes and proteomes, now we have the tools necessary for fast and precise elucidation of desirable crop traits. Here, we present an overview of high-throughput techniques used to analyze soybean responses to different abiotic (drought, flooding, cold stress, salinity, phosphate deficiency) and biotic (infections by F. oxysporum, cyst nematode, SMV) stress conditions at the level of the transcriptome (mRNAs and miRNAs) and the proteome.

A new short oligonucleotide-based strategy for the precursor-specific regulation of microRNA processing by dicer.

The precise regulation of microRNA (miRNA) biogenesis seems to be critically important for the proper functioning of all eukaryotic organisms. Even small changes in the levels of specific miRNAs can initiate pathological processes, including carcinogenesis. Accordingly, there is a great need to develop effective methods for the regulation of miRNA biogenesis and activity. In this study, we focused on the final step of miRNA biogenesis; i.e., miRNA processing by Dicer. To test our hypothesis that RNA molecules can function not only as Dicer substrates but also as Dicer regulators, we previously identified by SELEX a pool of RNA oligomers that bind to human Dicer. We found that certain of these RNA oligomers could selectively inhibit the formation of specific miRNAs. Here, we show that these specific inhibitors can simultaneously bind both Dicer and pre-miRNAs. These bifunctional riboregulators interfere with miRNA maturation by affecting pre-miRNA structure and sequestering Dicer. Based on these observations, we designed a set of short oligomers (12 nucleotides long) that were capable of influencing pre-miRNA processing in vitro, both in reactions involving recombinant human Dicer and in cytosolic extracts. We propose that the same strategy may be used to develop effective and selective regulators to control the production of any miRNA. Overall, our findings indicate that the interactions between pre-miRNAs and other RNAs may form very complex regulatory networks that modulate miRNA biogenesis and consequently gene expression.

Selection of RNA oligonucleotides that can modulate human dicer activity in vitro.

Human ribonuclease Dicer is an enzyme that excises small regulatory RNAs from perfectly or partially double-stranded RNA precursors. Although Dicer substrates and products have already been quite well characterized, our knowledge about cellular factors regulating the activity of this enzyme is still limited. To learn more about this problem, we attempted to determine whether RNA could function not only as a Dicer substrate but also as its regulator. To this end, we applied an in vitro selection method. We identified 120 RNA oligomers binding human Dicer. Sixteen of them were subjected to more detailed in vitro studies. We found that 6 out of 16 oligomers affected Dicer ability to digest pre-microRNAs (miRNAs), although most of them were cleaved by this enzyme. For the 6 most active oligomers the putative mechanism of Dicer inhibition was determined. Three oligomers were classified as typical competitive inhibitors and one as an allosteric inhibitor. The remaining 2 oligomers acted as selective inhibitors. They affected the production of 1 miRNA, whereas the formation of other miRNAs was hardly influenced. In general, the data obtained suggest that one can modulate the generation of specific miRNAs by using RNA oligomers. Moreover, we found that sequences similar to those of the selected oligomers can be found within the molecules composing human transcriptome.