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Among the magnetocaloric materials featuring first-order phase transitions (FOPT), FeRh is considered as a reference system to study the FOPT because it is a "simple" binary system with a CsCl structure exhibiting a large adiabatic temperature change. Recently, ab initio theory predicted that changes in the Fe/Rh stoichiometry in the vicinity of equiatomic composition strongly influence the FOPT characteristics. However, this theoretical prediction was not clearly verified experimentally. Here, we investigated the composition dependence of the transitional hysteresis in FeRh. It is shown that a Fe excess of only 1 at. % induces a ferromagnetic state in the whole temperature range (from 5 K up to T c ) for a minor portion of the sample (≈10%), while 5 at. % is enough to completely eliminate the FOPT. Element-specific X-ray magnetic circular dichroism (XMCD) measurements suggest that this ferromagnetic contribution arises from residual FeRh ferromagnetic regions. We attribute the formation of such domains to Fe antisite defects, as Mössbauer spectroscopy demonstrates the presence of Fe atoms located at the 1b (Rh) sites in the CsCl-type structure. As a consequence, compared with the equiatomic composition, the slightly Fe-rich sample exhibits completely different FOPT properties, influencing the magnetocaloric performances. Thus, our study sheds light on the origin of the remarkable stoichiometric sensitivity of the FOPT behavior in FeRh. These insights have broader implications for understanding FOPT dynamics and the role of residual ferromagnetic domains.

The transition toward a carbon-neutral society based on renewable energies goes hand in hand with the availability of energy-efficient technologies. Magnetocaloric cooling is a very promising refrigeration technology to fulfill this role regarding cryogenic gas liquefaction. However, the current reliance on highly resource critical, heavy rare-earth-based compounds as magnetocaloric material makes global usage unsustainable. Here, we aim to mitigate this limitation through the utilization of a multicaloric cooling concept, which uses the external stimuli of isotropic pressure and magnetic field to tailor and induce magnetostructural phase transitions associated with large caloric effects. In this study, La 0.7 Ce 0.3 Fe 11.6 Si 1.4 is used as a nontoxic, low-cost, low-criticality multiferroic material to explore the potential, challenges, and peculiarities of multicaloric cryocooling, achieving maximum isothermal entropy changes up to -28 J (kg K) -1 in the temperature range from 190 K down to 30 K. Thus, the multicaloric cooling approach offers an additional degree of freedom to tailor the phase transition properties and may lead to energy-efficient and environmentally friendly gas liquefaction based on designed-for-purpose, noncritical multiferroic materials.

Zero thermal expansion (ZTE) composites are typically designed by combining positive thermal expansion (PTE) with negative thermal expansion (NTE) materials acting as compensators and have many diverse applications, including in high-precision instrumentation and biomedical devices. La(Fe 1- x ,Si x )13-based compounds display several remarkable properties, such as giant magnetocaloric effect and very large NTE at room temperature. Both are linked via strong magnetovolume coupling, which leads to sharp magnetic and volume changes occurring simultaneously across first-order phase transitions; the abrupt nature of these changes makes them unsuitable as thermal expansion compensators. To make these materials more useful practically, the mechanisms controlling the temperature over which this transition occurs and the magnitude of contraction need to be controlled. In this work, ball-milling was used to decrease particles and crystallite sizes and increase the strain in LaFe 11.9 Mn 0.27 Si 1.29 H x alloys. Such size and strain tuning effectively broadened the temperature over which this transition occurs. The material's NTE operational temperature window was expanded, and its peak was suppressed by up to 85%. This work demonstrates that induced strain is the key mechanism controlling these materials' phase transitions. This allows the optimization of their thermal expansion toward room-temperature ZTE applications.

RNA-binding proteins play key roles in controlling gene expression in many organisms, but relatively few have been identified and characterised in detail in Gram-positive bacteria. Here, we globally analyse RNA-binding proteins in methicillin-resistant Staphylococcus aureus (MRSA) using two complementary biochemical approaches. We identify hundreds of putative RNA-binding proteins, many containing unconventional RNA-binding domains such as Rossmann-fold domains. Remarkably, more than half of the proteins containing helix-turn-helix (HTH) domains, which are frequently found in prokaryotic transcription factors, bind RNA in vivo. In particular, the CcpA transcription factor, a master regulator of carbon metabolism, uses its HTH domain to bind hundreds of RNAs near intrinsic transcription terminators in vivo. We propose that CcpA, besides acting as a transcription factor, post-transcriptionally regulates the stability of many RNAs., (© 2022. The Author(s).)

Extracellular vesicles (EVs) are released by cells from all kingdoms and represent one form of cell-cell interaction. This universal system of communication blurs cells type boundaries, offering an new avenue for pathogens to infect their hosts. EVs carry with them an arsenal of virulence factors that have been the focus of numerous studies. During the last years, the RNA content of EVs has also gained increasing attention, particularly in the context of infection. Secreted RNA in EVs (evRNA) from several bacterial pathogens have been characterised but the exact mechanisms promoting pathogenicity remain elusive. In this review, we evaluate the potential of such transcripts to directly interact with targets in infected cells and, by this, represent a novel angle of host cell manipulation during bacterial infection. This article is part of a Special Issue entitled: RNA and gene control in bacteria edited by Dr. M. Guillier and F. Repoila., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Post-transcriptional regulation of gene expression in cells is facilitated by formation of RNA-protein complexes (RNPs). While many methods to study eukaryotic (m)RNPs rely on purification of polyadenylated RNA, other important regulatory RNA classes or bacterial mRNA could not be investigated at the same depth. To overcome this limitation, we developed Phenol Toluol extraction (PTex), a novel and unbiased method for the purification of UV cross-linked RNPs in living cells. PTex is a fast (2-3 h) and simple protocol. The purification principle is solely based on physicochemical properties of cross-linked RNPs, enabling us to interrogate RNA-protein interactions system-wide and beyond poly(A) RNA from a variety of species and source material. Here, we are presenting an introduction of the underlying separation principles and give a detailed discussion of the individual steps as well as incorporation of PTex in high-throughput pipelines., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Protein-RNA interactions regulate all aspects of RNA metabolism and are crucial to the function of catalytic ribonucleoproteins. Until recently, the available technologies to capture RNA-bound proteins have been biased toward poly(A) RNA-binding proteins (RBPs) or involve molecular labeling, limiting their application. With the advent of organic-aqueous phase separation-based methods, we now have technologies that efficiently enrich the complete suite of RBPs and enable quantification of RBP dynamics. These flexible approaches to study RBPs and their bound RNA open up new research avenues for systems-level interrogation of protein-RNA interactions., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In recent years, hundreds of novel RNA-binding proteins (RBPs) have been identified, leading to the discovery of novel RNA-binding domains. Furthermore, unstructured or disordered low-complexity regions of RBPs have been identified to play an important role in interactions with nucleic acids. However, these advances in understanding RBPs are limited mainly to eukaryotic species and we only have limited tools to faithfully predict RNA-binders in bacteria. Here, we describe a support vector machine-based method, called TriPepSVM, for the prediction of RNA-binding proteins. TriPepSVM applies string kernels to directly handle protein sequences using tri-peptide frequencies. Testing the method in human and bacteria, we find that several RBP-enriched tri-peptides occur more often in structurally disordered regions of RBPs. TriPepSVM outperforms existing applications, which consider classical structural features of RNA-binding or homology, in the task of RBP prediction in both human and bacteria. Finally, we predict 66 novel RBPs in Salmonella Typhimurium and validate the bacterial proteins ClpX, DnaJ and UbiG to associate with RNA in vivo., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Recent methodological advances allowed the identification of an increasing number of RNA-binding proteins (RBPs) and their RNA-binding sites. Most of those methods rely, however, on capturing proteins associated to polyadenylated RNAs which neglects RBPs bound to non-adenylate RNA classes (tRNA, rRNA, pre-mRNA) as well as the vast majority of species that lack poly-A tails in their mRNAs (including all archea and bacteria). We have developed the Phenol Toluol extraction (PTex) protocol that does not rely on a specific RNA sequence or motif for isolation of cross-linked ribonucleoproteins (RNPs), but rather purifies them based entirely on their physicochemical properties. PTex captures RBPs that bind to RNA as short as 30 nt, RNPs directly from animal tissue and can be used to simplify complex workflows such as PAR-CLIP. Finally, we provide a global RNA-bound proteome of human HEK293 cells and the bacterium Salmonella Typhimurium.

RNA-binding proteins are important for core cellular processes such as mRNA transcription, splicing, transport, translation, and degradation. Recently, hundreds of novel RNA-binders have been identified in vivo in various organisms and cell types. We discuss the RNA interactome capture technique which enabled this boost in identifying new RNA-binding proteins in eukaryotes. A focus of this chapter, however, is the presentation of different challenges and problems that need to be addressed to be able to understand the conserved mRNA-bound proteomes from yeast to man.

RNA-binding proteins (RBPs) are key players in post-transcriptional regulation of gene expression in eukaryotic cells. To be able to unbiasedly identify RBPs in Saccharomyces cerevisiae, we developed a yeast RNA interactome capture protocol which employs RNA labeling, covalent UV crosslinking of RNA and proteins at 365nm wavelength (photoactivatable-ribonucleoside-enhanced crosslinking, PAR-CL) and finally purification of the protein-bound mRNA. The method can be easily implemented in common workflows and takes about 3days to complete. Next to a comprehensive explanation of the method, we focus on our findings about the choice of crosslinking in yeast and discuss the rationale of individual steps in the protocol., (Copyright © 2016. Published by Elsevier Inc.)

Post-transcriptional regulation of gene expression plays a critical role in almost all cellular processes. Regulation occurs mostly by RNA-binding proteins (RBPs) that recognise RNA elements and form ribonucleoproteins (RNPs) to control RNA metabolism from synthesis to decay. Recently, the repertoire of RBPs was significantly expanded owing to methodological advances such as RNA interactome capture. The newly identified RNA binders are involved in diverse biological processes and belong to a broad spectrum of protein families, many of them exhibiting enzymatic activities. This suggests the existence of an extensive crosstalk between RNA biology and other, in principle unrelated, cell functions such as intermediary metabolism. Unexpectedly, hundreds of new RBPs do not contain identifiable RNA-binding domains (RBDs), raising the question of how they interact with RNA. Despite the many functions that have been attributed to RNA, our understanding of RNPs is still mostly governed by a rather protein-centric view, leading to the idea that proteins have evolved to bind to and regulate RNA and not vice versa. However, RNPs formed by an RNA-driven interaction mechanism (RNA-determined RNPs) are abundant and offer an alternative explanation for the surprising lack of classical RBDs in many RNA-interacting proteins. Moreover, RNAs can act as scaffolds to orchestrate and organise protein networks and directly control their activity, suggesting that nucleic acids might play an important regulatory role in many cellular processes, including metabolism.

RNA-binding proteins (RBPs) exert a broad range of biological functions. To explore the scope of RBPs across eukaryotic evolution, we determined the in vivo RBP repertoire of the yeast Saccharomyces cerevisiae and identified 678 RBPs from yeast and additionally 729 RBPs from human hepatocytic HuH-7 cells. Combined analyses of these and recently published data sets define the core RBP repertoire conserved from yeast to man. Conserved RBPs harbour defined repetitive motifs within disordered regions, which display striking evolutionary expansion. Only 60% of yeast and 73% of the human RBPs have functions assigned to RNA biology or structural motifs known to convey RNA binding, and many intensively studied proteins surprisingly emerge as RBPs (termed 'enigmRBPs'), including almost all glycolytic enzymes, pointing to emerging connections between gene regulation and metabolism. Analyses of the mitochondrial hydroxysteroid dehydrogenase (HSD17B10) uncover the RNA-binding specificity of an enigmRBP.

RNA-protein complexes play pivotal roles in many central biological processes. Although methods based on high-throughput sequencing have advanced our ability to identify the specific RNAs bound by a particular protein, there is a need for precise and systematic ways to identify RNA interaction sites on proteins. We have developed an experimental and computational workflow combining photo-induced cross-linking, high-resolution mass spectrometry and automated analysis of the resulting mass spectra for the identification of cross-linked peptides, cross-linking sites and the cross-linked RNA oligonucleotide moieties of such RNA-binding proteins. The workflow can be applied to any RNA-protein complex of interest or to whole proteomes. We applied the approach to human and yeast mRNA-protein complexes in vitro and in vivo, demonstrating its powerful utility by identifying 257 cross-linking sites on 124 distinct RNA-binding proteins. The open-source software pipeline developed for this purpose, RNP(xl), is available as part of the OpenMS project.

Background: The Aquificales are a diverse group of thermophilic bacteria that thrive in terrestrial and marine hydrothermal environments. They can be divided into the families Aquificaceae, Desulfurobacteriaceae and Hydrogenothermaceae. Although eleven fully sequenced and assembled genomes are available, only little is known about this taxonomic order in terms of RNA metabolism., Results: In this work, we compare the available genomes, extend their protein annotation, identify regulatory sequences, annotate non-coding RNAs (ncRNAs) of known function, predict novel ncRNA candidates, show idiosyncrasies of the genetic decoding machinery, present two different types of transfer-messenger RNAs and variations of the CRISPR systems. Furthermore, we performed a phylogenetic analysis of the Aquificales based on entire genome sequences, and extended this by a classification among all bacteria using 16S rRNA sequences and a set of orthologous proteins.Combining several in silico features (e.g. conserved and stable secondary structures, GC-content, comparison based on multiple genome alignments) with an in vivo dRNA-seq transcriptome analysis of Aquifex aeolicus, we predict roughly 100 novel ncRNA candidates in this bacterium., Conclusions: We have here re-analyzed the Aquificales, a group of bacteria thriving in extreme environments, sharing the feature of a small, compact genome with a reduced number of protein and ncRNA genes. We present several classical ncRNAs and riboswitch candidates. By combining in silico analysis with dRNA-seq data of A. aeolicus we predict nearly 100 novel ncRNA candidates.

RNA-binding proteins (RBPs) determine RNA fate from synthesis to decay. Employing two complementary protocols for covalent UV crosslinking of RBPs to RNA, we describe a systematic, unbiased, and comprehensive approach, termed "interactome capture," to define the mRNA interactome of proliferating human HeLa cells. We identify 860 proteins that qualify as RBPs by biochemical and statistical criteria, adding more than 300 RBPs to those previously known and shedding light on RBPs in disease, RNA-binding enzymes of intermediary metabolism, RNA-binding kinases, and RNA-binding architectures. Unexpectedly, we find that many proteins of the HeLa mRNA interactome are highly intrinsically disordered and enriched in short repetitive amino acid motifs. Interactome capture is broadly applicable to study mRNA interactome composition and dynamics in varied biological settings., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Bacillus subtilis 6S-1 RNA binds to the housekeeping RNA polymerase (σ(A)-RNAP) and directs transcription of short 'product' RNAs (pRNAs). Here, we demonstrate that once newly synthesized pRNAs form a sufficiently stable duplex with 6S-1 RNA, a structural rearrangement is induced in cis, which involves base-pairing between sequences in the 5'-portion of the central bulge and nucleotides that become available as a result of pRNA invasion. The rearrangement decreases 6S-1 RNA affinity for σ(A)-RNAP. Among the pRNA length variants synthesized by σ(A)-RNAP (up to ∼14 nt), only the longer ones, such as 12-14-mers, form a duplex with 6S-1 RNA that is sufficiently long-lived to induce the rearrangement. Yet, an LNA (locked nucleic acid) 8-mer can induce the same rearrangement due to conferring increased duplex stability. We propose that an interplay of rate constants for polymerization (k(pol)), for pRNA:6S-1 RNA hybrid duplex dissociation (k(off)) and for the rearrangement (k(conf)) determines whether pRNAs dissociate or rearrange 6S-1 structure to trigger 6S-1 RNA release from σ(A)-RNAP. A bioinformatic screen suggests that essentially all bacterial 6S RNAs have the potential to undergo a pRNA-induced structural rearrangement.

By differential high-throughput RNA sequencing (dRNA-seq) we have identified "product RNAs" (pRNAs) as short as 8-12 nucleotides that are synthesized by Bacillus subtilis RNA polymerase (RNAP) in vivo using the regulatory 6S-1 RNA as template. The dRNA-seq data were confirmed by in vitro transcription experiments and Northern blotting. In our libraries, we were unable to detect statistically meaningful numbers of reads potentially representing pRNAs derived from 6S-2 RNA. However, pRNAs could be synthesized in vitro from 6S-2 RNA as template by the B. subtilis σ(A) RNAP. 6S-1 pRNA levels are low during exponential, increase in stationary, and burst during outgrowth from stationary phase, demonstrating that pRNA synthesis is a conserved regulatory mechanism, but a more dynamic and fine-tuning process than previously thought. Most pRNAs have a length of 8-15 nt, very few up to 24 nt. The average length of pRNAs tended to increase from stationary to outgrowth conditions. Synthesis of pRNA is initiated at C40 of 6S-1 RNA and U41 of 6S-2 RNA, yielding pRNAs with a 5'-terminal G or A residue, respectively. A B. subtilis 6S-1 RNA mutant strain encoding a pRNA with a 5'-terminal A residue showed the same relative distribution of ~14-nt pRNAs between the different growth states, but generally displayed lower pRNA levels than the reference strain encoding wild-type 6S-1 RNA. A ~two-fold lower affinity of the C40U mutant 6S-1 RNA towards σ(A) RNAP may have contributed to this reduction in pRNA levels. We infer that 6S-1 pRNA synthesis, although evolutionarily optimized for initiation with a +1G residue, is not primarily regulated at the transcription initiation level via growth phase-dependent variations in the cellular GTP pool.

Since off-target effects in non-viral siRNA delivery are quite common but not well understood, in this study various polymer-related effects observed in transfection studies were described and their mechanisms of toxicity were investigated. A variety of stably luciferase-expressing cell lines was compared concerning polymer-mediated effects after transfection with polyplexes of siRNA and poly(ethylene imine) (PEI) or poly(ethylene glycol)-grafted PEI (PEG-PEI). Cell viability, LDH release, gene expression profiles of apoptosis-related genes and promoter activation were investigated. Interestingly, PEG-PEI, which is generally better tolerated than PEI, was found to activate apoptosis in a cell line- and concentration-dependent manner. While both polymers showed sigmoidal dose-response of cell viability in L929 cells (IC(50)(PEI) = 6 μg/ml, IC(50)(PEG-PEI) = 11 μg/ml), H1299/Luc cells exhibited biphasic dose-response for PEG-PEI and stronger apoptosis at 2 μg/ml than at 20 μg/ml PEG-PEI, as shown in TUNEL assays. Gene expression profiling confirmed that H1299/Luc cells underwent apoptosis via thousand-fold activation of TNF receptor-associated factors. Additionally, it was demonstrated that NFkB-mediated CMV promoter activation in stably transfected cells can lead to increased target gene levels after transfection instead of siRNA-mediated knockdown. With these results, polymeric vectors were shown not to be inert substances. Therefore, alterations in gene expression caused by the delivery agent must be known to correctly interpret gene-silencing experiments, to understand the mechanisms of off-target effects, and most of all to further develop vectors with reduced side effects. Taking these observations into account, one established cell line was eventually identified to be suitable for RNAi experiments. As shown by these experiments, materials that have been used for many years can elicit unexpected off-target effects. Therefore, non-viral vectors must be screened for several levels of toxicity to make them promising candidates., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Here we describe a northern blot procedure that allows the detection of endogenous RNAs as small as approximately 14 nt in total RNA extracts from bacteria. RNAs that small and as part of total bacterial RNA extracts usually escape detection by northern blotting. The approach combines LNA probes 5'-digoxigenin-endlabeled for non-radioactive probe detection with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide-mediated chemical crosslinking of RNAs to nylon membranes, and necessitates the use of native PAGE either with the TBE or MOPS buffer system.

Evolutionary conserved mitochondrial nucleases are involved in programmed cell death and normal cell proliferation in lower and higher eukaryotes. The endo/exonuclease Nuc1p, also termed 'yeast Endonuclease G (EndoG)', is a member of this class of enzymes that differs from mammalian homologs by the presence of a 5'-3' exonuclease activity in addition to its broad spectrum endonuclease activity. However, this exonuclease activity is thought to be essential for a function of the yeast enzyme in DNA recombination and repair. Here we show that higher eukaryotes in addition to EndoG contain its paralog 'EXOG', a novel EndoG-like mitochondrial endo/exonuclease. We find that during metazoan evolution duplication of an ancestral nuclease gene obviously generated the paralogous EndoG- and EXOG-protein subfamilies in higher eukaryotes, thereby maintaining the full endo/exonuclease activity found in mitochondria of lower eukaryotes. We demonstrate that human EXOG is a dimeric mitochondrial enzyme that displays 5'-3' exonuclease activity and further differs from EndoG in substrate specificity. We hypothesize that in higher eukaryotes the complementary enzymatic activities of EndoG and EXOG probably together account for both, the lethal and vital functions of conserved mitochondrial endo/exonucleases.