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The single-stranded RNA genome of SARS-CoV-2 is highly structured. Numerous helical stem-loop structures interrupted by mismatch motifs are present in the functionally important 5'- and 3'-UTRs. These mismatches modulate local helical geometries and feature unusual arrays of hydrogen bonding donor and acceptor groups. However, their conformational and dynamical properties cannot be directly inferred from chemical probing and are difficult to predict theoretically. A mismatch motif (SL1-motif) consisting of three consecutive U•U base pairs is located in stem-loop 1 of the 3'-UTR. We combined NMR-spectroscopy and MD-simulations to investigate its structure and dynamics. All three U•U base pairs feature two direct hydrogen bonds and are as stable as Watson-Crick A:U base pairs. Plasmodium falciparum 25S rRNA contains a triple U•U mismatch motif (Pf-motif) differing from SL1-motif only with respect to the orientation of the two closing base pairs. Interestingly, while the geometry of the outer two U•U mismatches was identical in both motifs the preferred orientation of the central U•U mismatch was different. MD simulations and potassium ion titrations revealed that the potassium ion-binding mode to the major groove is connected to the different preferred geometries of the central base pair in the two motifs., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

We present the high-resolution structure of stem-loop 4 of the 5'-untranslated region (5_SL4) of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) genome solved by solution state nuclear magnetic resonance spectroscopy. 5_SL4 adopts an extended rod-like structure with a single flexible looped-out nucleotide and two mixed tandem mismatches, each composed of a G•U wobble base pair and a pyrimidine•pyrimidine mismatch, which are incorporated into the stem-loop structure. Both the tandem mismatches and the looped-out residue destabilize the stem-loop structure locally. Their distribution along the 5_SL4 stem-loop suggests a role of these non-canonical elements in retaining functionally important structural plasticity in particular with regard to the accessibility of the start codon of an upstream open reading frame located in the RNA's apical loop. The apical loop-although mostly flexible-harbors residual structural features suggesting an additional role in molecular recognition processes. 5_SL4 is highly conserved among the different variants of SARS-CoV-2 and can be targeted by small molecule ligands, which it binds with intermediate affinity in the vicinity of the non-canonical elements within the stem-loop structure., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Competing Interests: CK ist Berater von Innotope und hält eine Beteiligung an Innotope, einem Unternehmen, das Produkte zur Markierung stabiler RNA‐Isotope anbietet. Die übrigen Autoren erklären, dass sie keine konkurrierenden Interessen haben.

The synthetic neomycin-sensing riboswitch interacts with its cognate ligand neomycin as well as with the related antibiotics ribostamycin and paromomycin. Binding of these aminoglycosides induces a very similar ground state structure in the RNA, however, only neomycin can efficiently repress translation initiation. The molecular origin of these differences has been traced back to differences in the dynamics of the ligand:riboswitch complexes. Here, we combine five complementary fluorine based NMR methods to accurately quantify seconds to microseconds dynamics in the three riboswitch complexes. Our data reveal complex exchange processes with up to four structurally different states. We interpret our findings in a model that shows an interplay between different chemical groups in the antibiotics and specific bases in the riboswitch. More generally, our data underscore the potential of 19 F NMR methods to characterize complex exchange processes with multiple excited states., (© 2023 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Pseudouridine is the most frequently naturally occurring RNA modification, found in all classes of biologically functional RNAs. Compared to uridine, pseudouridine contains an additional hydrogen bond donor group and is therefore widely regarded as a structure stabilizing modification. However, the effects of pseudouridine modifications on the structure and dynamics of RNAs have so far only been investigated in a limited number of different structural contexts. Here, we introduced pseudouridine modifications into the U-turn motif and the adjacent U:U closing base pair of the neomycin-sensing riboswitch (NSR)-an extensively characterized model system for RNA structure, ligand binding, and dynamics. We show that the effects of replacing specific uridines with pseudouridines on RNA dynamics crucially depend on the exact location of the replacement site and can range from destabilizing to locally or even globally stabilizing. By using a combination of NMR spectroscopy, MD simulations and QM calculations, we rationalize the observed effects on a structural and dynamical level. Our results will help to better understand and predict the consequences of pseudouridine modifications on the structure and function of biologically important RNAs., (© 2023 Vögele et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The ongoing pandemic of the respiratory disease COVID-19 is caused by the SARS-CoV-2 (SCoV2) virus. SCoV2 is a member of the Betacoronavirus genus. The 30 kb positive sense, single stranded RNA genome of SCoV2 features 5'- and 3'-genomic ends that are highly conserved among Betacoronaviruses. These genomic ends contain structured cis-acting RNA elements, which are involved in the regulation of viral replication and translation. Structural information about these potential antiviral drug targets supports the development of novel classes of therapeutics against COVID-19. The highly conserved branched stem-loop 5 (SL5) found within the 5'-untranslated region (5'-UTR) consists of a basal stem and three stem-loops, namely SL5a, SL5b and SL5c. Both, SL5a and SL5b feature a 5'-UUUCGU-3' hexaloop that is also found among Alphacoronaviruses. Here, we report the extensive 1 H, 13 C and 15 N resonance assignment of the 37 nucleotides (nts) long sequence spanning SL5b and SL5c (SL5b + c), as basis for further in-depth structural studies by solution NMR spectroscopy., (© 2022. The Author(s).)

tRNAs are L-shaped RNA molecules of ~ 80 nucleotides that are responsible for decoding the mRNA and for the incorporation of the correct amino acid into the growing peptidyl-chain at the ribosome. They occur in all kingdoms of life and both their functions, and their structure are highly conserved. The L-shaped tertiary structure is based on a cloverleaf-like secondary structure that consists of four base paired stems connected by three to four loops. The anticodon base triplet, which is complementary to the sequence of the mRNA, resides in the anticodon loop whereas the amino acid is attached to the sequence CCA at the 3'-terminus of the molecule. tRNAs exhibit very stable secondary and tertiary structures and contain up to 10% modified nucleotides. However, their structure and function can also be maintained in the absence of nucleotide modifications. Here, we present the assignments of nucleobase resonances of the non-modified 77 nt tRNA Ile from the gram-negative bacterium Escherichia coli. We obtained assignments for all imino resonances visible in the spectra of the tRNA as well as for additional exchangeable and non-exchangeable protons and for heteronuclei of the nucleobases. Based on these assignments we could determine the chemical shift differences between modified and non-modified tRNA Ile as a first step towards the analysis of the effect of nucleotide modifications on tRNA's structure and dynamics., (© 2022. The Author(s).)

The SARS-CoV-2 virus is the cause of the respiratory disease COVID-19. As of today, therapeutic interventions in severe COVID-19 cases are still not available as no effective therapeutics have been developed so far. Despite the ongoing development of a number of effective vaccines, therapeutics to fight the disease once it has been contracted will still be required. Promising targets for the development of antiviral agents against SARS-CoV-2 can be found in the viral RNA genome. The 5'- and 3'-genomic ends of the 30 kb SCoV-2 genome are highly conserved among Betacoronaviruses and contain structured RNA elements involved in the translation and replication of the viral genome. The 40 nucleotides (nt) long highly conserved stem-loop 4 (5_SL4) is located within the 5'-untranslated region (5'-UTR) important for viral replication. 5_SL4 features an extended stem structure disrupted by several pyrimidine mismatches and is capped by a pentaloop. Here, we report extensive 1 H, 13 C, 15 N and 31 P resonance assignments of 5_SL4 as the basis for in-depth structural and ligand screening studies by solution NMR spectroscopy., (© 2021. The Author(s).)

The stem-loop (SL1) is the 5'-terminal structural element within the single-stranded SARS-CoV-2 RNA genome. It is formed by nucleotides 7-33 and consists of two short helical segments interrupted by an asymmetric internal loop. This architecture is conserved among Betacoronaviruses. SL1 is present in genomic SARS-CoV-2 RNA as well as in all subgenomic mRNA species produced by the virus during replication, thus representing a ubiquitous cis-regulatory RNA with potential functions at all stages of the viral life cycle. We present here the 1 H, 13 C and 15 N chemical shift assignment of the 29 nucleotides-RNA construct 5_SL1, which denotes the native 27mer SL1 stabilized by an additional terminal G-C base-pair., (© 2021. The Author(s).)

SARS-CoV-2 contains a positive single-stranded RNA genome of approximately 30 000 nucleotides. Within this genome, 15 RNA elements were identified as conserved between SARS-CoV and SARS-CoV-2. By nuclear magnetic resonance (NMR) spectroscopy, we previously determined that these elements fold independently, in line with data from in vivo and ex-vivo structural probing experiments. These elements contain non-base-paired regions that potentially harbor ligand-binding pockets. Here, we performed an NMR-based screening of a poised fragment library of 768 compounds for binding to these RNAs, employing three different 1 H-based 1D NMR binding assays. The screening identified common as well as RNA-element specific hits. The results allow selection of the most promising of the 15 RNA elements as putative drug targets. Based on the identified hits, we derive key functional units and groups in ligands for effective targeting of the RNA of SARS-CoV-2., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The SARS-CoV-2 (SCoV-2) virus is the causative agent of the ongoing COVID-19 pandemic. It contains a positive sense single-stranded RNA genome and belongs to the genus of Betacoronaviruses. The 5'- and 3'-genomic ends of the 30 kb SCoV-2 genome are potential antiviral drug targets. Major parts of these sequences are highly conserved among Betacoronaviruses and contain cis-acting RNA elements that affect RNA translation and replication. The 31 nucleotide (nt) long highly conserved stem-loop 5a (SL5a) is located within the 5'-untranslated region (5'-UTR) important for viral replication. SL5a features a U-rich asymmetric bulge and is capped with a 5'-UUUCGU-3' hexaloop, which is also found in stem-loop 5b (SL5b). We herein report the extensive 1 H, 13 C and 15 N resonance assignment of SL5a as basis for in-depth structural studies by solution NMR spectroscopy.

Phosphorothioates (PTs) are important chemical modifications of the RNA backbone where a single nonbridging oxygen of the phosphate is replaced with a sulfur atom. PT can stabilize RNAs by protecting them from hydrolysis and is commonly used as a tool to explore their function. It is, however, unclear what basic physical effects PT has on RNA stability and electronic structure. Here, we present molecular dynamics (MD) simulations, quantum mechanical (QM) calculations, and NMR spectroscopy measurements, exploring the effects of PT modifications in the structural context of the neomycin-sensing riboswitch (NSR). The NSR is the smallest biologically functional riboswitch with a well-defined structure stabilized by a U-turn motif. Three of the signature interactions of the U-turn: an H-bond, an anion-π interaction, and a potassium binding site; are formed by RNA phosphates, making the NSR an ideal model for studying how PT affects RNA structure and dynamics. By comparing with high-level QM calculations, we reveal the distinct physical properties of the individual interactions facilitated by the PT. The sulfur substitution, besides weakening the direct H-bond interaction, reduces the directionality of H-bonding while increasing its dispersion and induction components. It also reduces the induction and increases the dispersion component of the anion-π stacking. The sulfur force-field parameters commonly employed in the literature do not reflect these distinctions, leading to the unsatisfactory description of PT in simulations of the NSR. We show that it is not possible to accurately describe the PT interactions using one universal set of van der Waals sulfur parameters and provide suggestions for improving the force-field performance.

The current pandemic situation caused by the Betacoronavirus SARS-CoV-2 (SCoV2) highlights the need for coordinated research to combat COVID-19. A particularly important aspect is the development of medication. In addition to viral proteins, structured RNA elements represent a potent alternative as drug targets. The search for drugs that target RNA requires their high-resolution structural characterization. Using nuclear magnetic resonance (NMR) spectroscopy, a worldwide consortium of NMR researchers aims to characterize potential RNA drug targets of SCoV2. Here, we report the characterization of 15 conserved RNA elements located at the 5' end, the ribosomal frameshift segment and the 3'-untranslated region (3'-UTR) of the SCoV2 genome, their large-scale production and NMR-based secondary structure determination. The NMR data are corroborated with secondary structure probing by DMS footprinting experiments. The close agreement of NMR secondary structure determination of isolated RNA elements with DMS footprinting and NMR performed on larger RNA regions shows that the secondary structure elements fold independently. The NMR data reported here provide the basis for NMR investigations of RNA function, RNA interactions with viral and host proteins and screening campaigns to identify potential RNA binders for pharmaceutical intervention., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The neomycin sensing riboswitch is the smallest biologically functional RNA riboswitch, forming a hairpin capped with a U-turn loop-a well-known RNA motif containing a conserved uracil. It was shown previously that a U→C substitution of the eponymous conserved uracil does not alter the riboswitch structure due to C protonation at N3. Furthermore, cytosine is evolutionary permitted to replace uracil in other U-turns. Here, we use molecular dynamics simulations to study the molecular basis of this substitution in the neomycin sensing riboswitch and show that a structure-stabilizing monovalent cation-binding site in the wild-type RNA is the main reason for its negligible structural effect. We then use NMR spectroscopy to confirm the existence of this cation-binding site and to demonstrate its effects on RNA stability. Lastly, using quantum chemical calculations, we show that the cation-binding site is altering the electronic environment of the wild-type U-turn so that it is more similar to the cytosine mutant. The study reveals an amazingly complex and delicate interplay between various energy contributions shaping up the 3D structure and evolution of nucleic acids.

Up to now, the impact of the 1918 influenza pandemic on the German Imperial Army has remained surprisingly understudied. This paper is concerned with the two pandemic waves which afflicted the army during the last year of World War I and the attempts of prevention and treatment done by military doctors. Furthermore, it will be discussed how the disease was assessed by both military doctors and generals and to what extent German troops were affected by the pandemic. Finally it will be discussed if the pandemic changed the course of war.

Summary: Breastfeeding is considered to be the key variable for infant health. Consequently, UNICEF and the World Health Organization promote the beginning of breastfeeding within the first hour after birth and recommend to exclusively breastfeed the infant during the first six months. The origins of these modern breastfeeding campaigns can be traced back to the beginning of the twentieth century. Whereas high infant mortality rates traditionally were considered to be a matter of fate, declining birth rates towards the end of the nineteenth century raised fears about the nation's future and led to the emergence of an increasing infant welfare movement in Imperial Germany. As low breastfeeding rates were identified as a key factor behind the high infant mortality rates, the main objective of the infant care movement was to increase breastfeeding. The paper, therefore, focuses on medical breastfeeding recommendations and the attempts to popularize breastfeeding. At first, a sketch of medical doctrines will be presented, followed by a short survey of popular parental guidelines. Finally, two famous manuals of infant raising from the early twentieth century will be analysed in more detail. On the whole, the paper covers the period from the beginnings of social paediatrics at the beginning of the 20th century, the nutrition recommendations embedded into Nazi ideology during the Third Reich, until the declining breastfeeding ratios and the "feeding on demand"-movement in the 1970s as well as the ideological differences between West and East Germany during the Cold War.

Background: Aim was to analyze the association between local infection and amputation rate in patients with chronic critical limb ischemia (CLI) with or without successful revascularization., Patients and Methods: We performed a retrospective analysis of 56 consecutive patients with 57 critically ischemic legs seen at the University Hospital Bern. Patients with CLI were selected if ischemic lesions and follow-up of more than 2 months were documented. Infection was suggested when 2 of the following criterion were present: temperature > 37 degrees C, C-reactive protein > 50 mg/L, leukocytes > 10 x 10(3)/microliter ("2 of 3" criterion), or a putrid secretion was documented ("secretion" criterion)., Results: In patients with successful revascularization (n = 39), there was a significant shift from 10.3% major to 33.3% minor amputations (Chi Square p value = 0.014) as compared to patients without or with failed revascularization (n = 18) with 44.4% and 11.1% (Chi Square p value = 0.008), respectively. An infection was suggested in 22 of 53 limbs (41.5%) according to the "2 of 3" criterion, and 30 of 57 limbs (52.6%) satisfying the "secretion" criterion. Both criteria, were significantly more common in patients undergoing amputation as compared to patients without amputation (p = 0.001). Multiple lesions were more common in patients with major amputations (p = 0.026)., Conclusion: Successful revascularization effectively reduces major amputations and leads to healing of ischemic ulcers. Secondary foot infections are frequent. Infections are associated with a significantly higher rate of minor and major amputations, also in patients with successful revascularization, and should be treated adequately as well as in time with antibiotics.

This analysis assesses urban mortality change in Imperial Germany, when the country was going through a process of accelerated industrialization and urbanization. Urban mortality reached its peak after the middle of the century, thereafter urban mortality improved substantially. The largest cities, particularly in the highly industrialized western parts of the country, registered the strongest decline. A key element in this process was the reduction of mortality from gastrointestinal disorders, affecting almost exclusively infants. Therefore this analysis discusses the impact of selected public health strategies designed to fight high infant mortality rates. Special emphasis is placed on municipal milk supply and infant welfare centres.

Traditionally cities and towns in historical Europe were perceived as being particularly unhealthy. Terms like 'le handicap urbain' or 'urban penalty' have been introduced in order to emphasize the high death rates in the fast-growing industrial towns of nineteenth century Europe, which significantly exceeded the average rates for rural areas or the whole country. A rising population density was ideal for the transmission of the prevailing infectious diseases. This paper assesses urban and rural mortality change in Imperial Germany, when the country was going through a process of accelerated industrialization and urbanization. It provides an analysis of changes in age-, sex- and disease-specific mortality in urban and rural Prussia. In general, urban mortality in Germany reached its peak after the middle of the century, thereafter urban mortality improved substantially in relative as well as in absolute terms, the gap between urban and rural mortality narrowed and finally disappeared entirely. The largest cities registered the strongest decline in mortality. Obviously they had the potential to overcome the threats of disease or death, and became forerunners of improved health conditions in modern industrialized societies. An analysis of the mechanisms of mortality change in an urban environment during industrialization in the late nineteenth and early twentieth centuries can therefore serve as a paradigm for conditions in highly urbanized industrial societies.

Infant mortality in Imperial Germany started to decline in urban areas from the 1870s onwards, whereas national rates did not decrease before the beginning of the twentieth century. Therefore, key explanatory factors determining the levels and trends of infant mortality are investigated in an urban context. These include the decline of birth rates, the legitimacy status of infants, feeding practices, environmental conditions, and economic growth. Through a rising living standard and by creating a health-preserving environment, urban populations lost their traditional disadvantage in survival chances. This went so far that even high risk factors, such as the abandonment of breastfeeding, could be counterbalanced. In this sense, a study of past urban health conditions functions as a paradigm for the situation in industrialized societies.