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Overflow metabolism is a well-known phenomenon that describes the seemingly wasteful and incomplete substrate oxidation by aerobic cells, such as yeasts, bacteria, and mammalian cells, even when conditions allow for total combustion via respiration. This cellular response, triggered by an excess of C-source, has not yet been investigated in archaea. In this study, we conducted chemostat cultivations to compare the metabolic and physiological states of the thermoacidophilic archaeon Sulfolobus acidocaldarius under three conditions, each with gradually increasing nutrient stress. Our results show that S. acidocaldarius has different capacities for the uptake of the two C-sources, monosodium glutamate and glucose. A saturated tricarboxylic acid cycle at elevated nutrient concentrations affects the cell's ability to deplete its intermediates. This includes deploying additional cataplerotic pathways and the secretion of amino acids, notably valine, glycine, and alanine, while glucose is increasingly metabolized via glycogenesis. We did not observe the secretion of common fermentation products, like organic acids. Transcriptomic analysis indicated an upregulation of genes involved in fatty acid metabolism, suggesting the intracellular conservation of energy. Adapting respiratory enzymes under nutrient stress indicated high metabolic flexibility and robust regulatory mechanisms in this archaeon. This study enhances our fundamental understanding of the metabolism of S. acidocaldarius ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Sedlmayr, Széliová, De Kock, Gansemans, Van Nieuwerburgh, Peeters, Quehenberger, Zanghellini and Spadiut.)

Organization of archaeal chromatin combines bacterial, eukaryotic, and unique characteristics. Many archaeal lineages harbor a wide diversity of small and highly expressed nucleoid-associated proteins, which are involved in DNA structuring. In Sulfolobales, representing model organisms within the Crenarchaeota, Sul7d, Cren7, Sul10a, and Sul12a are well-characterized nucleoid-associated proteins. Here, we combine evidence that the Lrs14 family of DNA binders is part of the repertoire of nucleoid-associated proteins in Sulfolobales. Lrs14-encoding genes are widespread within genomes of different members of the Sulfolobales, typically encoded as four to nine homologs per genome. The Lrs14 proteins harbor a winged helix-turn-helix DNA-binding domain and are typified by a coiled-coil dimerization. They are characterized by distinct sequence- and structure-based features, including redox-sensitive motifs and residues targeted for posttranslational modification, allowing a further classification of the family into five conserved clusters. Lrs14-like proteins have unique DNA-organizing properties. By binding to the DNA nonsequence specifically and in a highly cooperative manner, with a slight preference for AT-rich promoter regions, they introduce DNA kinks and are able to affect transcription of adjacent transcription units either positively or negatively. Genes encoding Lrs14-type proteins display considerable differential expression themselves in response to various stress conditions, with certain homologs being specific to a particular stressor. Taken together, we postulate that members of the Lrs14 family can be considered nucleoid-associated proteins in Sulfolobales, combining a DNA-structuring role with a global gene expression role in response to stress conditions., (© 2024 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Obg is a widely conserved and essential GTPase in bacteria, which plays a central role in a large range of important cellular processes, such as ribosome biogenesis, DNA replication, cell division and bacterial persistence. Nevertheless, the exact function of Obg in these processes and the interactions it makes within the associated pathways remain largely unknown. Here, we identify the DNA-binding TrpD2 protein YbiB as an interactor of the Escherichia coli Obg (ObgE). We show that both proteins interact with high affinity in a peculiar biphasic fashion, and pinpoint the intrinsically disordered and highly negatively charged C-terminal domain of ObgE as a main driver for this interaction. Molecular docking and X-ray crystallography, together with site-directed mutagenesis, are used to map the binding site of this ObgE C-terminal domain within a highly positively charged groove on the surface of the YbiB homodimer. Correspondingly, ObgE efficiently inhibits the binding of DNA to YbiB, indicating that ObgE competes with DNA for binding in the positive clefts of YbiB. This study thus forms an important step for the further elucidation of the interactome and cellular role of the essential bacterial protein Obg., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

There are about 1500 genetic metabolic diseases. A small number of treatable diseases are diagnosed by newborn screening programs, which are continually being developed. However, most diseases can only be diagnosed based on clinical symptoms or metabolic findings. The main biological fluids used are urine, plasma and, in special situations, cerebrospinal fluid. In contrast to commonly used methods such as gas chromatography and high performance liquid chromatography mass spectrometry, ex vivo proton spectroscopy ( 1 H-NMR) is not yet used in routine clinical practice, although it has been recommended for more than 30 years. Automatic analysis and improved NMR technology have also expanded the applications used for the diagnosis of inborn errors of metabolism. We provide a mini-overview of typical applications, especially in urine but also in plasma, used to diagnose common but also rare genetic metabolic diseases with 1 H-NMR. The use of computer-assisted diagnostic suggestions can facilitate interpretation of the profiles. In a proof of principle, to date, 182 reports of 59 different diseases and 500 reports of healthy children are stored. The percentage of correct automatic diagnoses was 74%. Using the same 1 H-NMR profile-targeted analysis, it is possible to apply an untargeted approach that distinguishes profile differences from healthy individuals. Thus, additional conditions such as lysosomal storage diseases or drug interferences are detectable. Furthermore, because 1 H-NMR is highly reproducible and can detect a variety of different substance categories, the metabolomic approach is suitable for monitoring patient treatment and revealing additional factors such as nutrition and microbiome metabolism. Besides the progress in analytical techniques, a multiomics approach is most effective to combine metabolomics with, for example, whole exome sequencing, to also diagnose patients with nondetectable metabolic abnormalities in biological fluids. In this mini review we also provide our own data to demonstrate the role of NMR in a multiomics platform in the field of inborn errors of metabolism., (© 2022 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.)

Background: Handheld ultrasound devices present an opportunity for prehospital sonographic assessment of trauma, even in the hands of novice operators commonly found in military, maritime, or other austere environments. However, the reliability of such point-of-care ultrasound (POCUS) examinations by novices is rightly questioned. A common strategy being examined to mitigate this reliability gap is remote mentoring by an expert., Objectives: To assess the feasibility of utilizing POCUS in the hands of novice military or civilian emergency medicine service (EMS) providers, with and without the use of telementoring. To assess the mitigating or exacerbating effect telementoring may have on operator stress., Methods: Thirty-seven inexperienced physicians and EMTs serving as first responders in military or civilian EMS were randomized to receive or not receive telementoring during three POCUS trials: live model, Simbionix trainer, and jugular phantom. Salivary cortisol was obtained before and after the trial. Heart rate variability monitoring was performed throughout the trial., Results: There were no significant differences in clinical performance between the two groups. Iatrogenic complications of jugular venous catheterization were reduced by 26% in the telementored group (P < 0.001). Salivary cortisol levels dropped by 39% (P < 0.001) in the telementored group. Heart rate variability data also suggested mitigation of stress., Conclusions: Telementoring of POCUS tasks was not found to improve performance by novices, but findings suggest that it may mitigate caregiver stress.

In prokaryotes, transcription factors (TFs) are of uttermost importance for the regulation of gene expression. However, the majority of TFs are not characterized today, which hampers both the understanding of fundamental processes and the development of TF-based applications, such as biosensors, used in metabolic engineering, synthetic biology, diagnostics, etc. One way of analyzing TFs is through in vivo screening, enabling the study of TF-promoter interactions, ligand inducibility, and ligand specificity in a high-throughput fashion. Here, an approach is described for the selection and cloning of TF-promoter pairs, the development of a reporter system, and the measurement and analysis of fluorescent reporter assays. Furthermore, the importance of a suitable inducible plasmid system is illustrated together with prospective adaptations to modify a reporter system's output signal. The given approach can be used for the investigation of native, heterologous, or even artificially created TFs in Escherichia coli, and can be extended toward use in other microorganisms., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Emotions have an important survival function. Vast amounts of research have demonstrated how affect-related changes in physiology promote survival by effecting short-term and long-term changes in adaptive behavior. However, if emotions truly serve such an inherent function, they should be pervasive across species and be established early in life. Here, using electroencephalographic (EEG) brain activity we sought to characterize core neurophysiological features underlying affective function at the emergence of emotional expression [i.e., at the developmental age when human infants start to show reliable stimulus-elicited emotional states (4-6 months)]. Using an approach that eschews traditional EEG frequency band delineations (like theta, alpha), we demonstrate that negative emotional states induce a strong right hemispheric increase in the prominence of the resonant frequency (∼5-6 Hz) in the infant frontal EEG. Increased rightward asymmetry was strongly correlated with increased heart rate responses to emotionally negative states compared with neutral states. We conclude that functional frontal asymmetry is a key component of emotional processing and suggest that the rightward asymmetry in prominence of the resonant frequency during negative emotional states might reflect functional asymmetry in the central representation of anatomically driven asymmetry in the autonomic nervous system. Our findings indicate that the specific mode hallmarking emotional processing in the frontal cortex is established in parallel with the emergence of stable emotional states very early during development, despite the well known protracted maturation of frontal cortex., (Copyright © 2020 Bolinger et al.)

Background: Tension pneumothorax is a frequent cause of potentially preventable death. Tube thoracostomy (TT) can obviate death but is invasive and fraught with complications even in experienced hands. We assessed the utility of a remote international virtual network (RIVN) of specialized mentors to remotely guide military medical technicians (medics) using wireless informatics. Methods: Medics were randomized to insert TT in training mannequins (TraumaMan; Abacus ALS, Meadowbrook, Australia) supervised by RIVN or not. The RIVN consisted of trauma surgeons in Canada and Australia and a senior medic in Ohio. Medics wore a helmet-mounted wireless camera with laser pointer to confirm anatomy and two-way voice communication using commercial software (Skype ® ). Performance was measured through objective task completion (pass/fail) regarding safety during the procedure, proper location, and secure anchoring of the tube, in addition to remote mentor opinion and subjective debrief. Results: Fourteen medics attempted TT, seven mentored and seven not. The RIVN was functional and surgeons on either side of the globe had real-time communication with the mentees. TT placement was considered safe, successful, and secure in 100% of mentored ( n  = 7) procedures, although two (29%) received corrective remote guidance. All (100%) of the unmentored attempted and adequately secured the TT and were safe. However, only 71% ( n  = 5) completed the task successfully ( p  = 0.46). Participating medics subjectively felt remote telementoring (RTM) increased self-confidence (strong agreement mean 5/5 ± 0); confidence to perform field TT (agreement (4/5 ± 1); and decreased anxiety (strong agreement 5/5 ± 1). Subjectively, the remote mentors felt in 100% of the mentored procedures that "yes" they were able to assist the medics (1.86 ± 0.38), and in 71% ( n  = 5) felt "yes" they made TT safer (2.29 ± 0.49). Conclusions: RTM descriptively increased the success of TT placement and allowed for real-time troubleshooting from thousands of kilometers with a redundant capability. RTM was subjectively associated with high levels of satisfaction and self-reported self-confidence. Continued controlled and critical evaluation and refinement of telemedical techniques should continue. Trial Registration: ID ISRCTN/77929274.

Maturation of chloroplast psaA pre-mRNA from the green alga Chlamydomonas reinhardtii requires the trans-splicing of two split group II introns. Several nuclear-encoded trans-splicing factors are required for the correct processing of psaA mRNA. Among these is the recently identified Raa4 protein, which is involved in splicing of the tripartite intron 1 of the psaA precursor mRNA. Part of this tripartite group II intron is the chloroplast encoded tscA RNA, which is specifically bound by Raa4. Using Raa4 as bait in a combined tandem affinity purification and mass spectrometry approach, we identified core components of a multisubunit ribonucleoprotein complex, including three previously identified trans-splicing factors (Raa1, Raa3, and Rat2). We further detected tscA RNA in the purified protein complex, which seems to be specific for splicing of the tripartite group II intron. A yeast-two hybrid screen and co-immunoprecipitation identified chloroplast-localized Raa4-binding protein 1 (Rab1), which specifically binds tscA RNA from the tripartite psaA group II intron. The yeast-two hybrid system provides evidence in support of direct interactions between Rab1 and four trans-splicing factors. Our findings contribute to our knowledge of chloroplast multisubunit ribonucleoprotein complexes and are discussed in support of the generally accepted view that group II introns are the ancestors of the eukaryotic spliceosomal introns.

During trans-splicing of discontinuous organellar introns, independently transcribed coding sequences are joined together to generate a continuous mRNA. The chloroplast psaA gene from Chlamydomonas reinhardtii encoding the P(700) core protein of photosystem I (PSI) is split into three exons and two group IIB introns, which are both spliced in trans. Using forward genetics, we isolated a novel PSI mutant, raa4, with a defect in trans-splicing of the first intron. Complementation analysis identified the affected gene encoding the 112.4 kDa Raa4 protein, which shares no strong sequence identity with other known proteins. The chloroplast localization of the protein was confirmed by confocal fluorescence microscopy, using a GFP-tagged Raa4 fusion protein. RNA-binding studies showed that Raa4 binds specifically to domains D2 and D3, but not to other conserved domains of the tripartite group II intron. Raa4 may play a role in stabilizing folding intermediates or functionally active structures of the split intron RNA., (© 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.)