The structure of the pleiotropic transcription regulator CodY provides insight into its GTP-sensing mechanism.

GTP and branched-chain amino acids (BCAAs) are metabolic sensors that are indispensable for the determination of the metabolic status of cells. However, their molecular sensing mechanism remains unclear. CodY is a unique global transcription regulator that recognizes GTP and BCAAs as specific signals and affects expression of more than 100 genes associated with metabolism. Herein, we report the first crystal structures of the full-length CodY complex with sensing molecules and describe their functional states. We observed two different oligomeric states of CodY: a dimeric complex of CodY from Staphylococcus aureus with the two metabolites GTP and isoleucine, and a tetrameric form (apo) of CodY from Bacillus cereus Notably, the tetrameric state shows in an auto-inhibitory manner by blocking the GTP-binding site, whereas the binding sites of GTP and isoleucine are clearly visible in the dimeric state. The GTP is located at a hinge site between the long helical region and the metabolite-binding site. Together, data from structural and electrophoretic mobility shift assay analyses improve understanding of how CodY senses GTP and operates as a DNA-binding protein and a pleiotropic transcription regulator.

Structural insights into the dimer-tetramer transition of FabI from Bacillus anthracis.

Enoyl-ACP reductase (ENR, also known as FabI) has received considerable interest as an anti-bacterial target due to its essentiality in fatty acid synthesis. All the FabI structures reported to date, regardless of the organism, are composed of homo-tetramers, except for two structures: Bacillus cereus and Staphylococcus aureus FabI (bcFabI and saFabI, respectively), which have been reported as dimers. However, the reason for the existence of the dimeric form in these organisms and the biological meaning of dimeric and tetrameric forms of FabI are ambiguous. Herein, we report the high-resolution crystal structure of a dimeric form of Bacillus anthracis FabI (baFabI) and the crystal structures of tetrameric forms of baFabI in the apo state and in complex with NAD+ and with NAD+-triclosan, at 1.7 Å, 1.85 Å, 1.96 Å, and 1.95 Å, respectively. Interestingly, we found that baFabI with a His6-tag at its C-terminus exists as a dimer, whereas untagged-baFabI exists as a tetramer. The His6-tag may block the dimer-tetramer transition, since baFabI has relatively short-length amino acids (255LG256) after the 310-helix η7 compared to those of FabI of other organisms. The dimeric form of baFabI is catalytically inactive, because the α-helix α5 occupies the NADH-binding site. During the process of dimer-tetramer transition, this α5 helix rotates about 55° toward the tetramer interface and the active site is established. Therefore, tetramerization of baFabI is required for cofactor binding and catalytic activity.

Analysis of Chain Branch of Polyolefins by a New Proton NMR Approach.

The crystallinity of polyethylene, which significantly affects the properties of the polymer, is quite sensitive to the concentration of its branches. Thus, it is necessary to estimate branch concentration with reasonable accuracy. Currently, (13)C NMR and gel permeation chromatography-Fourier transform infrared spectroscopy are widely-used analysis methods for the analysis of branch concentration. Despite several advantages, these methods sometimes have limitations. For instance, the preparation of samples for (13)C- NMR is tedious because high-concentration samples are required and the time for analysis is greater than 12 h. To more efficiently estimate the branch concentration of polyethylene, we developed a new high-field (1)H NMR method with an improved peak resolution by employing (1) homonuclear decoupling and (2) 2D heteronuclear correlation. The new method was observed to significantly reduce the experimental time to ∼ 30 min; furthermore, sample preparation was relatively simple because the method did not require high-concentration samples.

Processing of A-form ssDNA by cryptic RNase H fold exonuclease PF2046.

RNase H fold protein PF2046 of Pyrococcus furiosus is a 3'-5' ssDNA exonuclease that cleaves after the second nucleotide from the 3' end of ssDNA and prefers poly-dT over poly-dA as a substrate. In our crystal structure of PF2046 complexed with an oligonucleotide of four thymidine nucleotides (dT4), PF2046 accommodates dT4 tightly in a groove and imposes steric hindrance on dT4 mainly by Phe220 such that dT4 assumes the A-form. As poly-dA prefer B-form due to the stereochemical restrictions, the A-form ssDNA binding by PF2046 should disfavor the processing of poly-dA. Phe220 variants display reduced activity toward poly-dA and the A-form appears to be a prerequisite for the processing by PF2046.

Structural Insights into the Rrp4 Subunit from the Crystal Structure of the Thermoplasma acidophilum Exosome.

The exosome multiprotein complex plays a critical role in RNA processing and degradation. This system governs the regulation of mRNA quality, degradation in the cytoplasm, the processing of short noncoding RNA, and the breakdown of RNA fragments. We determined two crystal structures of exosome components from Thermoplasma acidophilum (Taci): one with a resolution of 2.3 Å that reveals the central components (TaciRrp41 and TaciRrp42), and another with a resolution of 3.5 Å that displays the whole exosome (TaciRrp41, TaciRrp42, and TaciRrp4). The fundamental exosome structure revealed the presence of a heterodimeric complex consisting of TaciRrp41 and TaciRrp42. The structure comprises nine subunits, with TaciRrp41 and TaciRrp42 arranged in a circular configuration, while TaciRrp4 is located at the apex. The RNA degradation capabilities of the TaciRrp4:41:42 complex were verified by RNA degradation assays, consistent with prior findings in other archaeal exosomes. The resemblance between archaeal exosomes and bacterial PNPase suggests a common mechanism for RNA degradation. Despite sharing comparable topologies, the surface charge distributions of TaciRrp4 and other archaea structures are surprisingly distinct. Different RNA breakdown substrates may be responsible for this variation. These newfound structural findings enhance our comprehension of RNA processing and degradation in biological systems.

Development of carbon nanoparticles-based soluble solid-phase immune sensor for the quantitative diagnosis of inflammation.

Quantitative immunodiagnosis is one of the most commonly used methods for in vitro diagnostics. Various bioanalytical methods have been developed to quantitatively diagnose immune analytes; however, they require blood dilution pretreatment, reaction mixing, complicated experimental steps, and can cause diagnostic errors due to the hook effect. To address this issue, we introduced a simple immunoassay based on carbon nanoparticles (CNPs). The assay was designed to have high flexibility for use in various in vitro diagnostic devices by constructing a soluble solid-phase immune sensor with high solubility using antibody-conjugated CNPs and polymer materials. Excellent performance was achieved using a free-antibody system with dual calibration. To verify the performance of this method with high reliability, canine C-reactive protein was selected as the immune analyte. Interestingly, our method efficiently mitigated the hook effect with outstanding performance in a one-step reaction without blood dilution or reaction mixing. The detection range of the target can be effectively controlled using free antibodies. Therefore, our CNP-based immunodiagnosis method may advance the commercialization of point-of-care immune biosensors.

EPRS1 Controls the TGF-ß Signaling Pathway via Interaction with TßRI in Hepatic Stellate Cell.

Glutamyl-prolyl-tRNA synthetase 1 (EPRS1) is known to associated with fibrosis through its catalytic activity to produce prolyl-tRNA. Although its catalytic inhibitor halofuginone (HF) has been known to inhibit the TGF-ß pathway as well as to reduce prolyl-tRNA production for the control of fibrosis, the underlying mechanism how EPRS1 regulates the TGF-ß pathway was not fully understood. Here, we show a noncatalytic function of EPRS1 in controlling the TGF-ß pathway and hepatic stellate cell activation via its interaction with TGF-ß receptor I (TßRI). Upon stimulation with TGF-ß, EPRS1 is phosphorylated by TGF-ß-activated kinase 1 (TAK1), leading to its dissociation from the multi-tRNA synthetase complex and subsequent binding with TßRI. This interaction increases the association of TßRI with SMAD2/3 while decreases that of TßRI with SMAD7. Accordingly, EPRS1 stabilizes TßRI by preventing the ubiquitin-mediated degradation of TßRI. HF disrupts the interaction between EPRS1 and TßRI, and reduces TßRI protein levels, leading to inhibition of the TGF-ß pathway. In conclusion, this work suggests the novel function of EPRS1 involved in the development of fibrosis by regulating the TGF-ß pathway and the antifibrotic effects of HF by controlling both of EPRS1 functions.

Control of fibrosis with enhanced safety via asymmetric inhibition of prolyl-tRNA synthetase 1.

Prolyl-tRNA synthetase 1 (PARS1) has attracted much interest in controlling pathologic accumulation of collagen containing high amounts of proline in fibrotic diseases. However, there are concerns about its catalytic inhibition for potential adverse effects on global protein synthesis. We developed a novel compound, DWN12088, whose safety was validated by clinical phase 1 studies, and therapeutic efficacy was shown in idiopathic pulmonary fibrosis model. Structural and kinetic analyses revealed that DWN12088 binds to catalytic site of each protomer of PARS1 dimer in an asymmetric mode with different affinity, resulting in decreased responsiveness at higher doses, thereby expanding safety window. The mutations disrupting PARS1 homodimerization restored the sensitivity to DWN12088, validating negative communication between PARS1 promoters for the DWN12088 binding. Thus, this work suggests that DWN12088, an asymmetric catalytic inhibitor of PARS1 as a novel therapeutic agent against fibrosis with enhanced safety.

Severe acute respiratory syndrome coronavirus 2 variants-Possibility of universal vaccine design: A review.

Both novel and conventional vaccination strategies have been implemented worldwide since the onset of coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Despite various medical advances in the treatment and prevention of the spread of this contagious disease, it remains a major public health threat with a high mortality rate. As several lethal SARS-CoV-2 variants continue to emerge, the development of several vaccines and medicines, each with certain advantages and disadvantages, is underway. Additionally, many modalities are at various stages of research and development or clinical trials. Here, we summarize emerging SARS-CoV-2 variants, including delta, omicron, and "stealth omicron," as well as available oral drugs for COVID-19. We also discuss possible antigen candidates other than the receptor-binding domain protein for the development of a universal COVID-19 vaccine. The present review will serve as a helpful resource for future vaccine and drug development to combat COVID-19.

Automatic and quantitative measurement of alveolar bone level in OCT images using deep learning.

We propose a method to automatically segment the periodontal structures of the tooth enamel and the alveolar bone using convolutional neural network (CNN) and to measure quantitatively and automatically the alveolar bone level (ABL) by detecting the cemento-enamel junction and the alveolar bone crest in optical coherence tomography (OCT) images. The tooth enamel and the alveolar bone regions were automatically segmented using U-Net, Dense-UNet, and U2-Net, and the ABL was quantitatively measured as the distance between the cemento-enamel junction and the alveolar bone crest using image processing. The mean distance difference (MDD) measured by our suggested method ranged from 0.19 to 0.22 mm for the alveolar bone crest (ABC) and from 0.18 to 0.32 mm for the cemento-enamel junction (CEJ). All CNN models showed the mean absolute error (MAE) of less than 0.25 mm in the x and y coordinates and greater than 90% successful detection rate (SDR) at 0.5 mm for both the ABC and the CEJ. The CNN models showed high segmentation accuracies in the tooth enamel and the alveolar bone regions, and the ABL measurements at the incisors by detected results from CNN predictions demonstrated high correlation and reliability with the ground truth in OCT images.

O-GlcNAc modification of leucyl-tRNA synthetase 1 integrates leucine and glucose availability to regulate mTORC1 and the metabolic fate of leucine.

All living organisms have the ability to sense nutrient levels to coordinate cellular metabolism. Despite the importance of nutrient-sensing pathways that detect the levels of amino acids and glucose, how the availability of these two types of nutrients is integrated is unclear. Here, we show that glucose availability regulates the central nutrient effector mTORC1 through intracellular leucine sensor leucyl-tRNA synthetase 1 (LARS1). Glucose starvation results in O-GlcNAcylation of LARS1 on residue S1042. This modification inhibits the interaction of LARS1 with RagD GTPase and reduces the affinity of LARS1 for leucine by promoting phosphorylation of its leucine-binding site by the autophagy-activating kinase ULK1, decreasing mTORC1 activity. The lack of LARS1 O-GlcNAcylation constitutively activates mTORC1, supporting its ability to sense leucine, and deregulates protein synthesis and leucine catabolism under glucose starvation. This work demonstrates that LARS1 integrates leucine and glucose availability to regulate mTORC1 and the metabolic fate of leucine.

Protocol for improving diffraction quality of leucyl-tRNA synthetase 1 with methylation and post-crystallization soaking and cooling in cryoprotectants.

Leucyl-tRNA synthetase 1 (LARS1) synthesizes Leu-tRNALeu for protein synthesis and plays an important role in mTORC1 activation by sensing intracellular leucine concentrations. Here, we describe a protocol for the purification, reductive methylation, binding affinity measurement by microscale thermophoresis, T i value measurement by Tycho, and post-crystallization soaking and cooling in cryoprotectants to improve crystallization of LARS1. Collectively, this allowed us to build the RagD binding domain, which was shown to be a dynamic region of LARS1 refractory to crystallization. For complete details on the use and execution of this protocol, please refer to Kim et al. (2021).

Hopeless tooth and less posterior occlusion is related to a greater risk of low handgrip strength: A population-based cross-sectional study.

The effect of severely compromised teeth on masticatory function has not been properly evaluated in previous studies, as they were often considered equivalent to the healthy tooth or excluded as if absent in the dentition. Hopeless teeth, which refer to non-salvageable teeth that require extraction, can interfere with masticatory function. As posterior occlusion is directly related to the masticatory function, we evaluated pairs opposing posterior teeth (POPs) that reflect the arrangement as well as the number of remaining posterior teeth. This study investigated the relationship of a hopeless tooth to handgrip strength according to POPs in the elderly. This cross-sectional study used data from the Korea National Health and Nutrition Examination Survey (KNHANES). Among the data of 23,466 participants from 2015 to 2018, participants aged 60 years or older (n = 4,729) were included. In males with POPs scores of 0-7, considered poor posterior occlusion, the association with low handgrip strength persisted in the multivariate logistic regression model adjusted for all confounding variables. The odds ratio (OR) in the absence of hopeless teeth (OR = 1.91, 95% CI: 1.02-3.59) increased in the presence of a hopeless tooth (OR = 2.78, 95% CI: 1.42-5.47). Even with POPs scores of 8-11, considered good posterior occlusion, the association was significantly high in the presence of a hopeless tooth (OR = 2.82, 95% CI: 1.06-7.52). In females, the association disappeared in adjusted models. The fewer pairs of natural posterior teeth with occlusion, the greater the risk of low handgrip strength. Dentition containing hopeless teeth increases the risk of low handgrip strength, even in dentition with sufficient posterior occlusion. Preserving the posterior teeth in a healthy condition through personal oral hygiene and regular dental management is essential for maintaining components of physical function such as handgrip strength.

Incorporating a real-time automatic alerting system based on electronic medical records could improve rapid response systems: a retrospective cohort study.

BACKGROUND: Rapid response systems (RRSs) are essential components of patient safety systems; however, limited evidence exists regarding their effectiveness and optimal structures. We aimed to assess the activation patterns and outcomes of RRS implementation with/without a real-time automatic alerting system (AAS) based on electronic medical records (EMRs). METHODS: We retrospectively analyzed clinical data of patients for whom the RRS was activated in the surgical wards of a tertiary university hospital. We compared the code rate, in-hospital mortality, unplanned intensive care unit (ICU) admission, and other clinical outcomes before and after applying RRS and AAS as follows: pre-RRS (January 2013-July 2015), RRS without AAS (August 2015-November 2016), and RRS with AAS (December 2016-December 2017). RESULTS: In-hospital mortality per 1000 admissions decreased from 15.1 to 12.9 after RRS implementation (p < 0.001). RRS activation per 1000 admissions increased from 14.4 to 26.3 after AAS implementation. The severity of patients' condition calculated using the modified early warning score increased from 2.5 (± 2.1) in the RRS without AAS to 3.6 (± 2.1) (p < 0.001) in the RRS with AAS. The total and preventable code rates and in-hospital mortality rates were comparable between the RRS implementation periods without/with AAS. ICU duration and mortality results improved in patients with RRS activation and unplanned ICU admission. The data of RRS non-activated group remained unaltered during the study. CONCLUSIONS: Real-time AAS based on EMRs might help identify unstable patients. Early detection and intervention with RRS may improve patient outcomes.

Structural Insights into a Bifunctional Peptide Methionine Sulfoxide Reductase MsrA/B Fusion Protein from Helicobacter pylori.

Methionine sulfoxide reductase (Msr) is a family of enzymes that reduces oxidized methionine and plays an important role in the survival of bacteria under oxidative stress conditions. MsrA and MsrB exist in a fusion protein form (MsrAB) in some pathogenic bacteria, such as Helicobacter pylori (Hp), Streptococcus pneumoniae, and Treponema denticola. To understand the fused form instead of the separated enzyme at the molecular level, we determined the crystal structure of HpMsrABC44S/C318S at 2.2 Å, which showed that a linker region (Hpiloop, 193-205) between two domains interacted with each HpMsrA or HpMsrB domain via three salt bridges (E193-K107, D197-R103, and K200-D339). Two acetate molecules in the active site pocket showed an sp2 planar electron density map in the crystal structure, which interacted with the conserved residues in fusion MsrABs from the pathogen. Biochemical and kinetic analyses revealed that Hpiloop is required to increase the catalytic efficiency of HpMsrAB. Two salt bridge mutants (D193A and E199A) were located at the entrance or tailgate of Hpiloop. Therefore, the linker region of the MsrAB fusion enzyme plays a key role in the structural stability and catalytic efficiency and provides a better understanding of why MsrAB exists in a fused form.

Insights into the structure of mature streptavidin C1 from Streptomyces cinnamonensis reveal the self-binding of the extension C-terminal peptide to biotin-binding sites.

The members of the avidin protein family are well known for their high affinity towards d-biotin and their structural stability. These properties make avidins a valuable tool for various biotechnological applications. In the present study, two avidin-like biotin-binding proteins (named streptavidin C1 and C2) from Streptomyces cinnamonensis were newly identified while exploring antifungal proteins against Fusarium oxysporum f. sp. cucumerinum. Streptavidin C1 reveals a low correlation (a sequence identity of approximately 64%) with all known streptavidins, whereas streptavidin C2 shares a sequence identity of approximately 94% with other streptavidins. Here, the crystal structures of streptavidin C1 in the mature form and in complex with biotin at 2.1 and 2.5 Šresolution, respectively, were assessed. The overall structures present similar tetrameric features with D 2 symmetry to other (strept)avidin structures. Interestingly, the long C-terminal region comprises a short α-helix (C-Lid; residues 169-179) and an extension C-terminal peptide (ECP; residues 180-191) which stretches into the biotin-binding sites of the same monomer. This ECP sequence (-180VTSANPPAS188-) is a newly defined biotin-binding site, which reduces the ability to bind to (strept)avidin family proteins. The novel streptavidin C1 could help in the development of an engineered tetrameric streptavidin with reduced biotin-binding capacity as well as other biomaterial tools.

Leucine-sensing mechanism of leucyl-tRNA synthetase 1 for mTORC1 activation.

Leucyl-tRNA synthetase 1 (LARS1) mediates activation of leucine-dependent mechanistic target of rapamycin complex 1 (mTORC1) as well as ligation of leucine to its cognate tRNAs, yet its mechanism of leucine sensing is poorly understood. Here we describe leucine binding-induced conformational changes of LARS1. We determine different crystal structures of LARS1 complexed with leucine, ATP, and a reaction intermediate analog, leucyl-sulfamoyl-adenylate (Leu-AMS), and find two distinct functional states of LARS1 for mTORC1 activation. Upon leucine binding to the synthetic site, H251 and R517 in the connective polypeptide and 50FPYPY54 in the catalytic domain change the hydrogen bond network, leading to conformational change in the C-terminal domain, correlating with RagD association. Leucine binding to LARS1 is increased in the presence of ATP, further augmenting leucine-dependent interaction of LARS1 and RagD. Thus, this work unveils the structural basis for leucine-dependent long-range communication between the catalytic and RagD-binding domains of LARS1 for mTORC1 activation.

Structural Basis for the Antibiotic Resistance of Eukaryotic Isoleucyl-tRNA Synthetase.

Pathogenic aminoacyl-tRNA synthetases (ARSs) are attractive targets for anti-infective agents because their catalytic active sites are different from those of human ARSs. Mupirocin is a topical antibiotic that specifically inhibits bacterial isoleucy-ltRNA synthetase (IleRS), resulting in a block to protein synthesis. Previous studies on Thermus thermophilus IleRS indicated that mupirocin-resistance of eukaryotic IleRS is primarily due to differences in two amino acids, His581 and Leu583, in the active site. However, without a eukaryotic IleRS structure, the structural basis for mupirocin-resistance of eukaryotic IleRS remains elusive. Herein, we determined the crystal structure of Candida albicans IleRS complexed with Ile-AMP at 2.9 Å resolution. The largest difference between eukaryotic and prokaryotic IleRS enzymes is closure of the active site pocket by Phe55 in the HIGH loop; Arg410 in the CP core loop; and the second Lys in the KMSKR loop. The Ile-AMP product is lodged in a closed active site, which may restrict its release and thereby enhance catalytic efficiency. The compact active site also prevents the optimal positioning of the 9-hydroxynonanoic acid of mupirocin and plays a critical role in resistance of eukaryotic IleRS to anti-infective agents.

Effectiveness of a daytime rapid response system in hospitalized surgical ward patients.

BACKGROUND: Clinical deteriorations during hospitalization are often preventable with a rapid response system (RRS). We aimed to investigate the effectiveness of a daytime RRS for surgical hospitalized patients. METHODS: A retrospective cohort study was conducted in 20 general surgical wards at a 1,779-bed University hospital from August 2013 to July 2017 (August 2013 to July 2015, pre-RRS-period; August 2015 to July 2017, post-RRS-period). The primary outcome was incidence of cardiopulmonary arrest (CPA) when the RRS was operating. The secondary outcomes were the incidence of total and preventable cardiopulmonary arrest, in-hospital mortality, the percentage of "do not resuscitate" orders, and the survival of discharged CPA patients. RESULTS: The relative risk (RR) of CPA per 1,000 admissions during RRS operational hours (weekdays from 7 AM to 7 PM) in the post-RRS-period compared to the pre-RRS-period was 0.53 (95% confidence interval [CI], 0.25 to 1.13; P=0.099) and the RR of total CPA regardless of RRS operating hours was 0.76 (95% CI, 0.46 to 1.28; P=0.301). The preventable CPA after RRS implementation was significantly lower than that before RRS implementation (RR, 0.31; 95% CI, 0.11 to 0.88; P=0.028). There were no statistical differences in in-hospital mortality and the survival rate of patients with in-hospital cardiac arrest. Do-not-resuscitate decisions significantly increased during after RRS implementation periods compared to pre-RRS periods (RR, 1.91; 95% CI, 1.40 to 2.59; P<0.001). CONCLUSIONS: The day-time implementation of the RRS did not significantly reduce the rate of CPA whereas the system effectively reduced the rate of preventable CPA during periods when the system was operating.

Monothiol and dithiol glutaredoxin-1 from Clostridium oremlandii: identification of domain-swapped structures by NMR, X-ray crystallography and HDX mass spectrometry.

Protein dimerization or oligomerization resulting from swapping part of the protein between neighboring polypeptide chains is known to play a key role in the regulation of protein function and in the formation of protein aggregates. Glutaredoxin-1 from Clostridium oremlandii (cGrx1) was used as a model to explore the formation of multiple domain-swapped conformations, which were made possible by modulating several hinge-loop residues that can form a pivot for domain swapping. Specifically, two alternative domain-swapped structures were generated and analyzed using nuclear magnetic resonance (NMR), X-ray crystallography, circular-dichroism spectroscopy and hydrogen/deuterium-exchange (HDX) mass spectrometry. The first domain-swapped structure (ß3-swap) was formed by the hexameric cGrx1-cMsrA complex. The second domain-swapped structure (ß1-swap) was formed by monothiol cGrx1 (C16S) alone. In summary, the first domain-swapped structure of an oxidoreductase in a hetero-oligomeric complex is presented. In particular, a single point mutation of a key cysteine residue to serine led to the formation of an intramolecular disulfide bond, as opposed to an intermolecular disulfide bond, and resulted in modulation of the underlying free-energy landscape of protein oligomerization.