Thermodynamic characterization of an engineered tetracycline-binding riboswitch

Riboswitches reflect a novel concept in gene regulation that is particularly suited for technological adaptation. Therefore, we characterized thermodynamically the ligand binding properties of a synthetic, tetracycline (tc)-binding RNA aptamer, which regulates gene expression in a dose-dependent manner when inserted into the untranslated region of an mRNA. In vitro, one molecule of tc is bound by one molecule of partially pre-structured and conformationally homogeneous apo-RNA. The dissociation constant of 770 pM, as determined by fluorimetry, is the lowest reported so far for a small molecule-binding RNA aptamer. Additional calorimetric analysis of RNA point mutants and tc derivatives identifies functional groups crucial for the interaction and including their respective enthalpic and entropic contributions we can propose detailed structural and functional roles for certain groups. The conclusions are consistent with mutational analyses in vivo and support the hypothesis that tc-binding reinforces the structure of the RNA aptamer, preventing the scanning ribosome from melting it efficiently

Tetracycline aptamer-controlled regulation of pre-mRNA splicing in yeast

Splicing of pre-mRNA is a critical step in mRNA maturation and disturbances cause several genetic disorders. We apply the synthetic tetracycline (tc)-binding riboswitch to establish a gene expression system for conditional tc-dependent control of pre-mRNA splicing in yeast. Efficient regulation is obtained when the aptamer is inserted close to the 5′splice site (SS) with the consensus sequence of the SS located within the aptamer stem. Structural probing indicates limited spontaneous cleavage within this stem in the absence of the ligand. Addition of tc leads to tightening of the stem and the whole aptamer structure which probably prevents recognition of the 5′SS. Combination of more then one aptamer-regulated intron increases the extent of regulation leading to highly efficient conditional gene expression systems. Our findings highlight the potential of direct RNA–ligand interaction for regulation of gene expression

Mechanistic insights into an engineered riboswitch: a switching element which confers riboswitch activity

While many different RNA aptamers have been identified that bind to a plethora of small molecules only very few are capable of acting as engineered riboswitches. Even for aptamers binding the same ligand large differences in their regulatory potential were observed. We address here the molecular basis for these differences by using a set of unrelated neomycin-binding aptamers. UV melting analyses showed that regulating aptamers are thermally stabilized to a significantly higher degree upon ligand binding than inactive ones. Regulating aptamers show high ligand-binding affinity in the low nanomolar range which is necessary but not sufficient for regulation. NMR data showed that a destabilized, open ground state accompanied by extensive structural changes upon ligand binding is important for regulation. In contrast, inactive aptamers are already pre-formed in the absence of the ligand. By a combination of genetic, biochemical and structural analyses, we identified a switching element responsible for destabilizing the ligand free state without compromising the bound form. Our results explain for the first time the molecular mechanism of an engineered riboswitch

Conformational dynamics of the tetracycline-binding aptamer

The conformational dynamics induced by ligand binding to the tetracycline-binding aptamer is monitored via stopped-flow fluorescence spectroscopy and time-correlated single photon counting experiments. The fluorescence of the ligand is sensitive to changes within the tertiary structure of the aptamer during and after the binding process. In addition to the wild-type aptamer, the mutants A9G, A13U and A50U are examined, where bases important for regulation are changed to inhibit the aptamer’s function. Our results suggest a very fast two-step-mechanism for the binding of the ligand to the aptamer that can be interpreted as a binding step followed by a reorganization of the aptamer to accommodate the ligand. Binding to the two direct contact points A13 and A50 was found to occur in the first binding step. The exchange of the structurally important base A9 for guanine induces an enormous deceleration of the overall binding process, which is mainly rooted in an enhancement of the back reaction of the first binding step by several orders of magnitude. This indicates a significant loss of tertiary structure of the aptamer in the absence of the base A9, and underlines the importance of pre-organization on the overall binding process of the tetracycline-binding aptamer

A fast and efficient translational control system for conditional expression of yeast genes

A new artificial regulatory system for essential genes in yeast is described. It prevents translation of target mRNAs upon tetracycline (tc) binding to aptamers introduced into their 5'UTRs. Exploiting direct RNA–ligand interaction renders auxiliary protein factors unnecessary. Therefore, our approach is strain independent and not susceptible to interferences by heterologous expressed regulatory proteins. We use a simple PCR-based strategy, which allows easy tagging of any target gene and the level of gene expression can be adjusted due to various tc aptamer-regulated promoters. As proof of concept, five differently expressed genes were targeted, two of which could not be regulated previously. In all cases, adding tc completely prevented growth and, as shown for Nop14p, rapidly abolished de novo protein synthesis providing a powerful tool for conditional regulation of yeast gene expression

Neutral sphingomyelinase mediates the co-morbidity trias of alcohol abuse, major depression and bone defects

Mental disorders are highly comorbid and occur together with physical diseases, which are often considered to arise from separate pathogenic pathways. We observed in alcohol-dependent patients increased serum activity of neutral sphingomyelinase. A genetic association analysis in 456,693 volunteers found associations of haplotypes of SMPD3 coding for NSM-2 (NSM) with alcohol consumption, but also with affective state, and bone mineralisation. Functional analysis in mice showed that NSM controls alcohol consumption, affective behaviour, and their interaction by regulating hippocampal volume, cortical connectivity, and monoaminergic responses. Furthermore, NSM controlled bone–brain communication by enhancing osteocalcin signalling, which can independently supress alcohol consumption and reduce depressive behaviour. Altogether, we identified a single gene source for multiple pathways originating in the brain and bone, which interlink disorders of a mental–physical co-morbidity trias of alcohol abuse—depression/anxiety—bone disorder. Targeting NSM and osteocalcin signalling may, thus, provide a new systems approach in the treatment of a mental–physical co-morbidity trias

Difficult tracheal intubation in neonates and infants. NEonate and Children audiT of Anaesthesia pRactice IN Europe (NECTARINE): a prospective European multicentre observational study

Background: Neonates and infants are susceptible to hypoxaemia in the perioperative period. The aim of this study was to analyse interventions related to anaesthesia tracheal intubations in this European cohort and identify their clinical consequences. Methods: We performed a secondary analysis of tracheal intubations of the European multicentre observational trial (NEonate and Children audiT of Anaesthesia pRactice IN Europe [NECTARINE]) in neonates and small infants with difficult tracheal intubation. The primary endpoint was the incidence of difficult intubation and the related complications. The secondary endpoints were the risk factors for severe hypoxaemia attributed to difficult airway management, and 30 and 90 day outcomes. Results: Tracheal intubation was planned in 4683 procedures. Difficult tracheal intubation, defined as two failed attempts of direct laryngoscopy, occurred in 266 children (271 procedures) with an incidence (95% confidence interval [CI]) of 5.8% (95% CI, 5.1e6.5). Bradycardia occurred in 8% of the cases with difficult intubation, whereas a significant decrease in oxygen saturation (SpO2<90% for 60 s) was reported in 40%. No associated risk factors could be identified among comorbidities, surgical, or anaesthesia management. Using propensity scoring to adjust for confounders, difficult anaesthesia tracheal intubation did not lead to an increase in 30 and 90 day morbidity or mortality. Conclusions: The results of the present study demonstrate a high incidence of difficult tracheal intubation in children less than 60 weeks post-conceptual age commonly resulting in severe hypoxaemia. Reassuringly, the morbidity and mortality at 30 and 90 days was not increased by the occurrence of a difficult intubation event. Clinical trial registration: NCT02350348

Difficult tracheal intubation in neonates and infants. NEonate and Children audiT of Anaesthesia pRactice IN Europe (NECTARINE): a prospective European multicentre observational study

BACKGROUND: Neonates and infants are susceptible to hypoxaemia in the perioperative period. The aim of this study was to analyse interventions related to anaesthesia tracheal intubations in this European cohort and identify their clinical consequences. METHODS: We performed a secondary analysis of tracheal intubations of the European multicentre observational trial (NEonate and Children audiT of Anaesthesia pRactice IN Europe [NECTARINE]) in neonates and small infants with difficult tracheal intubation. The primary endpoint was the incidence of difficult intubation and the related complications. The secondary endpoints were the risk factors for severe hypoxaemia attributed to difficult airway management, and 30 and 90 day outcomes. RESULTS: Tracheal intubation was planned in 4683 procedures. Difficult tracheal intubation, defined as two failed attempts of direct laryngoscopy, occurred in 266 children (271 procedures) with an incidence (95% confidence interval [CI]) of 5.8% (95% CI, 5.1–6.5). Bradycardia occurred in 8% of the cases with difficult intubation, whereas a significant decrease in oxygen saturation (SpO2<90% for 60 s) was reported in 40%. No associated risk factors could be identified among co-morbidities, surgical, or anaesthesia management. Using propensity scoring to adjust for confounders, difficult anaesthesia tracheal intubation did not lead to an increase in 30 and 90 day morbidity or mortality. CONCLUSIONS: The results of the present study demonstrate a high incidence of difficult tracheal intubation in children less than 60 weeks post-conceptual age commonly resulting in severe hypoxaemia. Reassuringly, the morbidity and mortality at 30 and 90 days was not increased by the occurrence of a difficult intubation event

Correction to: Two years later: Is the SARS-CoV-2 pandemic still having an impact on emergency surgery? An international cross-sectional survey among WSES members

Background: The SARS-CoV-2 pandemic is still ongoing and a major challenge for health care services worldwide. In the first WSES COVID-19 emergency surgery survey, a strong negative impact on emergency surgery (ES) had been described already early in the pandemic situation. However, the knowledge is limited about current effects of the pandemic on patient flow through emergency rooms, daily routine and decision making in ES as well as their changes over time during the last two pandemic years. This second WSES COVID-19 emergency surgery survey investigates the impact of the SARS-CoV-2 pandemic on ES during the course of the pandemic. Methods: A web survey had been distributed to medical specialists in ES during a four-week period from January 2022, investigating the impact of the pandemic on patients and septic diseases both requiring ES, structural problems due to the pandemic and time-to-intervention in ES routine. Results: 367 collaborators from 59 countries responded to the survey. The majority indicated that the pandemic still significantly impacts on treatment and outcome of surgical emergency patients (83.1% and 78.5%, respectively). As reasons, the collaborators reported decreased case load in ES (44.7%), but patients presenting with more prolonged and severe diseases, especially concerning perforated appendicitis (62.1%) and diverticulitis (57.5%). Otherwise, approximately 50% of the participants still observe a delay in time-to-intervention in ES compared with the situation before the pandemic. Relevant causes leading to enlarged time-to-intervention in ES during the pandemic are persistent problems with in-hospital logistics, lacks in medical staff as well as operating room and intensive care capacities during the pandemic. This leads not only to the need for triage or transferring of ES patients to other hospitals, reported by 64.0% and 48.8% of the collaborators, respectively, but also to paradigm shifts in treatment modalities to non-operative approaches reported by 67.3% of the participants, especially in uncomplicated appendicitis, cholecystitis and multiple-recurrent diverticulitis. Conclusions: The SARS-CoV-2 pandemic still significantly impacts on care and outcome of patients in ES. Well-known problems with in-hospital logistics are not sufficiently resolved by now; however, medical staff shortages and reduced capacities have been dramatically aggravated over last two pandemic years

Comprehensive Fragment Screening of the SARS-CoV-2 Proteome Explores Novel Chemical Space for Drug Development

12 pags., 4 figs., 3 tabs.SARS-CoV-2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including anti-virals. Within the international Covid19-NMR consortium, we have identified binders targeting the RNA genome of SCoV2. We established protocols for the production and NMR characterization of more than 80 % of all SCoV2 proteins. Here, we performed an NMR screening using a fragment library for binding to 25 SCoV2 proteins and identified hits also against previously unexplored SCoV2 proteins. Computational mapping was used to predict binding sites and identify functional moieties (chemotypes) of the ligands occupying these pockets. Striking consensus was observed between NMR-detected binding sites of the main protease and the computational procedure. Our investigation provides novel structural and chemical space for structure-based drug design against the SCoV2 proteome.Work at BMRZ is supported by the state of Hesse. Work in Covid19-NMR was supported by the Goethe Corona Funds, by the IWBEFRE-program 20007375 of state of Hesse, the DFG through CRC902: “Molecular Principles of RNA-based regulation.” and through infrastructure funds (project numbers: 277478796, 277479031, 392682309, 452632086, 70653611) and by European Union’s Horizon 2020 research and innovation program iNEXT-discovery under grant agreement No 871037. BY-COVID receives funding from the European Union’s Horizon Europe Research and Innovation Programme under grant agreement number 101046203. “INSPIRED” (MIS 5002550) project, implemented under the Action “Reinforcement of the Research and Innovation Infrastructure,” funded by the Operational Program “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014–2020) and co-financed by Greece and the EU (European Regional Development Fund) and the FP7 REGPOT CT-2011-285950—“SEE-DRUG” project (purchase of UPAT’s 700 MHz NMR equipment). The support of the CERM/CIRMMP center of Instruct-ERIC is gratefully acknowledged. This work has been funded in part by a grant of the Italian Ministry of University and Research (FISR2020IP_02112, ID-COVID) and by Fondazione CR Firenze. A.S. is supported by the Deutsche Forschungsgemeinschaft [SFB902/B16, SCHL2062/2-1] and the Johanna Quandt Young Academy at Goethe [2019/AS01]. M.H. and C.F. thank SFB902 and the Stiftung Polytechnische Gesellschaft for the Scholarship. L.L. work was supported by the French National Research Agency (ANR, NMR-SCoV2-ORF8), the Fondation de la Recherche Médicale (FRM, NMR-SCoV2-ORF8), FINOVI and the IR-RMN-THC Fr3050 CNRS. Work at UConn Health was supported by grants from the US National Institutes of Health (R01 GM135592 to B.H., P41 GM111135 and R01 GM123249 to J.C.H.) and the US National Science Foundation (DBI 2030601 to J.C.H.). Latvian Council of Science Grant No. VPP-COVID-2020/1-0014. National Science Foundation EAGER MCB-2031269. This work was supported by the grant Krebsliga KFS-4903-08-2019 and SNF-311030_192646 to J.O. P.G. (ITMP) The EOSC Future project is co-funded by the European Union Horizon Programme call INFRAEOSC-03-2020—Grant Agreement Number 101017536. Open Access funding enabled and organized by Projekt DEALPeer reviewe