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Three 819 knots in closed-loop strands of different lengths (~20, 23, and 26 nm) were used to experimentally assess the consequences of knot tightness at the molecular level. Through the use of ¹H NMR, diffusion-ordered spectroscopy (DOSY), circular dichroism (CD), collision-induced dissociation mass spectrometry (CID-MS) and molecular dynamics (MD) simulations on the different-sized knots, we find that the structure, dynamics, and reactivity of the molecular chains are dramatically affected by the tightness of the knotting. The tautness of entanglement causes differences in conformation, enhances the expression of topological chirality, weakens covalent bonds, inhibits decomplexation events, and changes absorption properties. Understanding the effects of tightening nanoscale knots may usefully inform the design of knotted and entangled molecular materials. [ABSTRACT FROM AUTHOR]

Individuals generally form their unique memories from shared experiences, yet the neural representational mechanisms underlying this subjectiveness of memory are poorly understood. The current study addressed this important question from the cross-subject neural representational perspective, leveraging a large functional magnetic resonance imaging dataset (n = 415) of a face--name associative memory task. We found that individuals' memory abilities were predicted by their synchronization to the group-averaged, canonical trial-by-trial activation level and, to a lesser degree, by their similarity to the group-averaged representational patterns during encoding. More importantly, the memory content shared between pairs of participants could be predicted by their shared local neural activation pattern, particularly in the angular gyrus and ventromedial prefrontal cortex, even after controlling for differences in memory abilities. These results uncover neural representational mechanisms for individualized memory and underscore the constructive nature of episodic memory. [ABSTRACT FROM AUTHOR]

Quinone molecules are intracellular electron-transport carriers, as well as critical intra- and extracellular signals. However, transcriptional regulation of quinone signaling and its molecular basis are poorly understood. Here, we identify a thiol-stress-sensing regulator YodB family transcriptional regulator as a central component of quinone stress response of Staphylococcus aureus, which we have termed the quinone-sensing and response repressor (QsrR). We also identify and confirm an unprecedented quinone-sensing mechanism based on the S-quinonization of the essential residue Cys-5. Structural characterizations of the QsrR-DNA and QsrR-menadione complexes further reveal that the covalent association of menadione directly leads to the release of QsrR from operator DNA following a 10° rigid-body rotation as well as a 9-Å elongation between the dimeric subunits. The molecular level characterization of this quinone-sensing transcriptional regulator provides critical insights into quinone-mediated gene regulation in human pathogens. [ABSTRACT FROM AUTHOR]

Hemodynamic shear stress stimulates a number of intracellular events that both regulate vessel structure and influence development of vascular pathologies. The precise molecular mechanisms by which endothelial cells transduce this mechanical stimulus into intracellular biochemical response have not been established. Here, we show that mechanical perturbation of the plasma membrane leads to ligand-independent conformational transitions in a G protein-coupled receptor (GPCR). By using time-resolved fluorescence microscopy and GPCR conformation-sensitive FRET we found that stimulation of endothelial cells with fluid shear stress, hypotonic stress, or membrane fluidizing agent leads to a significant increase in activity of bradykinin B2 GPCR in endothelial cells. The GPCR conformational dynamics was detected by monitoring redistribution of GPCRs between inactive and active conformations in a single endothelial cell under fluid shear stress in real time. We show that this response can be blocked by a B2-selective antagonist. Our data demonstrate that changes in cell membrane tension and membrane fluidity affect conformational dynamics of GPCRs. Therefore, we suggest that GPCRs are involved in mediating primary mechanochemical signal transduction in endothelial cells. We anticipate our experiments to be a starting point for more sophisticated studies of the effects of changes in lipid bilayer environment on GPCR conformational dynamics. Furthermore, because GPCRs are a major target of drug development, a detailed characterization of mechanochemical signaling via the GPCR pathway will be relevant for the development of new antiatherosclerosis drugs. [ABSTRACT FROM AUTHOR]

Acute myeloid leukemia (AML) is an aging-related and heterogeneous hematopoietic malignancy. In this study, a total of 1,474 newly diagnosed AML patients with RNA sequencing data were enrolled, and targeted or whole exome sequencing data were obtained in 94% cases. The correlation of aging-related factors including age and clonal hematopoiesis (CH), gender, and genomic/transcriptomic profiles (gene fusions, genetic mutations, and gene expression networks or pathways) was systematically analyzed. Overall, AML patients aged 60 y and older showed an apparently dismal prognosis. Alongside age, the frequency of gene fusions defined in the World Health Organization classification decreased, while the positive rate of gene mutations, especially CH-related ones, increased. Additionally, the number of genetic mutations was higher in gene fusion--negative (GF-) patients than those with GF. Based on the status of CH- and myelodysplastic syndromes (MDS)-related mutations, three mutant subgroups were identified among the GF-AML cohort, namely, CH-AML, CH-MDS-AML, and other GF-AML. Notably, CH-MDS-AML demonstrated a predominance of elderly and male cases, cytopenia, and significantly adverse clinical outcomes. Besides, gene expression networks including HOXA/B, platelet factors, and inflammatory responses were most striking features associated with aging and poor prognosis in AML. Our work has thus unraveled the intricate regulatory circuitry of interactions among different age, gender, and molecular groups of AML. [ABSTRACT FROM AUTHOR]

The constant emergence of COVID-19 variants reduces the effectiveness of existing vaccines and test kits. Therefore, it is critical to identify conserved structures in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes as potential targets for variant-proof diagnostics and therapeutics. However, the algorithms to predict these conserved structures, which simultaneously fold and alignmultiple RNA homologs, scale at best cubically with sequence length and are thus infeasible for coronaviruses, which possess the longest genomes (~30,000 nt) among RNA viruses. As a result, existing efforts on modeling SARS-CoV-2 structures resort to single-sequence folding as well as local folding methods with short window sizes, which inevitably neglect longrange interactions that are crucial in RNA functions. Here we present LinearTurboFold, an efficient algorithm for folding RNA homologs that scales linearly with sequence length, enabling unprecedented global structural analysis on SARS-CoV-2. Surprisingly, on a group of SARS-CoV-2 and SARS-related genomes, LinearTurboFold's purely in silico prediction not only is close to experimentally guided models for local structures, but also goes far beyond them by capturing the end-to-end pairs between 5' and 3' untranslated regions (UTRs) (~29,800 nt apart) that match perfectly with a purely experimental work. Furthermore, LinearTurboFold identifies undiscovered conserved structures and conserved accessible regions as potential targets for designing efficient andmutation-insensitive small-molecule drugs, antisense oligonucleotides, small interfering RNAs (siRNAs), CRISPR-Cas13 guide RNAs, and RT-PCR primers. LinearTurboFold is a general technique that can also be applied to other RNA viruses and fulllength genome studies and will be a useful tool in fighting the current and future pandemics. [ABSTRACT FROM AUTHOR]

How to illuminate dark matter has become the foremost open question in fundamental science nowadays, which is of great significance in understanding the laws of nature. Exploring exotic interactions beyond the standard model is one of the essential approaches to searching for dark matter particles. Although it has been explored in a variety of lab-scale and tabletop-scale setups over the past years, no such interactions have been observed, and improving the sensitivity significantly becomes of paramount importance, but challenging. Here, we formulate the conception of a spin-mechanical quantum chip compatible with scalable on-chip detectors. Utilizing the prototype chip realized by the integration of a mechanical resonator and a diamond with single nitrogen vacancy at the microscale, the constraints of spin-velocity-dependent interactions have been improved by two orders of magnitude, where there is no evidence for new bosons in the force range below 100 nm, i.e., in the rest-mass window of 2–10 electronvolts. Based on the proof-of-principle experiment, this promising chip can be scaled up to meet the requirements of searching for exotic interactions at preeminent sensitivity. Low-cost and high-yield chip-scale setups will accelerate the process of dark matter exploration, providing a path toward on-chip fundamental physics experiments. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, uses an RNA-dependent RNA polymerase along with several accessory factors to replicate its genome and transcribe its genes. Nonstructural protein (nsp) 13 is a helicase required for viral replication. Here, we found that nsp13 ligates iron, in addition to zinc, when purified anoxically. Using inductively coupled plasma mass spectrometry, UV-visible absorption, EPR, and Mössbauer spectroscopies, we characterized nsp13 as an iron--sulfur (Fe--S) protein that ligates an Fe4S4 cluster in the treble-clef metal-binding site of its zinc-binding domain. The Fe--S cluster in nsp13 modulates both its binding to the template RNA and its unwinding activity. Exposure of the protein to the stable nitroxide TEMPOL oxidizes and degrades the cluster and drastically diminishes unwinding activity. Thus, optimal function of nsp13 depends on a labile Fe--S cluster that is potentially targetable for COVID-19 treatment. [ABSTRACT FROM AUTHOR]

Both chronic obstructive pulmonary disease (COPD) and asthma are severe respiratory diseases. Bitter receptor-mediated bronchodilation is a potential therapy for asthma, but the mechanism underlying the agonistic relaxation of airway smooth muscle (ASM) is not well defined. By exploring the ASM relaxation mechanism of bitter substances, we observed that pretreatment with the bitter substances nearly abolished the methacholine (MCh)-induced increase in the ASM cell (ASMC) calcium concentration, thereby suppressing the calcium-induced contraction release. The ASM relaxation was significantly inhibited by simultaneous deletion of three Gαt proteins, suggesting an interaction between Tas2R and AChR signaling cascades in the relaxation process. Biochemically, the Gαt released by Tas2R activation complexes with AChR and blocks the Gαq cycling of AChR signal transduction. More importantly, a bitter substance, kudinoside A, not only attenuates airway constriction but also significantly inhibits pulmonary inflammation and tissue remodeling in COPD rats, indicating its modulation of additional Gαq-associated pathological processes. Thus, our results suggest that Tas2R activation may be an ideal strategy for halting multiple pathological processes of COPD. [ABSTRACT FROM AUTHOR]

Sonic Hedgehog (Shh) is a key signaling molecule that plays important roles in various developmental processes in mammals. Although the signal transduction pathway activated by Shh is well understood, the regulation of its secretion remains unclear. Newly synthesized Shh is imported into the endoplasmic reticulum (ER), where it undergoes a series of posttranslational modifications to produce the mature lipid-modified amino-terminal fragment. Here, we have analyzed the molecular mechanisms that mediate secretion of the N-terminal fragment of Shh (ShhN). We found that the Cardin-Weintraub (CW) motif in Shh is necessary and sufficient for ER-to-Golgi transport of ShhN. Mechanistic analyses revealed that a cargo receptor, Surfeit locus protein 4 (SURF4), interacts directly with the CWmotif of ShhN to regulate packaging of ShhN into COPII vesicles. ShhN and SURF4 interact with each other at the ER and separate from each other after entering the Golgi. The CW motif is known to interact with proteoglycans (PGs) that are predominantly synthesized at the Golgi. Interestingly, we found that PGs compete with SURF4 to bind ShhN and that inhibiting synthesis of PGs causes defects in export of ShhN from the trans Golgi network (TGN). SURF4 and PG maturation are also important for intracellular traffic of full length Shh in mammalian cells. Our study suggests a SURF4-to-PG relay mechanism that mediates the sorting and secretion of Shh, providing insight into the biosynthetic trafficking of Shh. [ABSTRACT FROM AUTHOR]

FTO demethylates internal N6-methyladenosine (m6A) and N6,2'-Odimethyladenosine (m6Am; at the cap +1 position) in mRNA, m6A and m6Am in snRNA, and N1-methyladenosine (m1A) in tRNA in vivo, and in vitro evidence supports that it can also demethylate N6-methyldeoxyadenosine (6 mA), 3-methylthymine (3mT), and 3-methyluracil (m3U). However, it remains unclear how FTO variously recognizes and catalyzes these diverse substrates. Here we demonstrate--in vitro and in vivo--that FTO has extensive demethylation enzymatic activity on both internal m6A and cap m6Am. Considering that 6mA, m6A, and m6Am all share the same nucleobase, we present a crystal structure of human FTO bound to 6mAmodified ssDNA, revealing the molecular basis of the catalytic demethylation of FTO toward multiple RNA substrates. We discovered that (i) N6-methyladenine is the most favorable nucleobase substrate of FTO, (ii) FTO displays the same demethylation activity toward internal m6A andm6Am in the same RNA sequence, suggesting that the substrate specificity of FTO primarily results from the interaction of residues in the catalytic pocket with the nucleobase (rather than the ribose ring), and (iii) the sequence and the tertiary structure of RNA can affect the catalytic activity of FTO. Our findings provide a structural basis for understanding the catalytic mechanism through which FTO demethylates its multiple substrates and pave the way forward for the structure-guided design of selective chemicals for functional studies and potential therapeutic applications. [ABSTRACT FROM AUTHOR]

Seed weight (SW), which is one of the three major factors influencing grain yield, has been widely accepted as a complex trait that is controlled by polygenes, particularly in polyploid crops. Brassica napus L., which is the second leading crop source for vegetable oil around the world, is a tetraploid (4×) species. In the present study, we identified a major quantitative trait locus (QTL) on chromosome A9 of rapeseed in which the genes for SW and silique length (SL) were colocated. By fine mapping and association analysis, we uncovered a 165-bp deletion in the auxin-response factor 18 (ARF18) gene associated with increased SW and SL. ARF18 encodes an auxinresponse factor and shows inhibitory activity on downstream auxin genes. This 55-aa deletion prevents ARF18 fromforming homodimers, in turn resulting in the loss of binding activity. Furthermore, reciprocal crossing has shown that this QTL affects SW by maternal effects. Transcription analysis has shown that ARF18 regulates cell growth in the silique wall by acting via an auxin-response pathway. Together, our results suggest that ARF18 regulates silique wall development and determines SW via maternal regulation. In addition, our study reveals the first (to our knowledge) QTL in rapeseed and may provide insights into gene cloning involving polyploid crops. [ABSTRACT FROM AUTHOR]

To date, efforts to switch the cofactor specificity of oxidoreductases from nicotinamide adenine dinucleotide phosphate (NADPH) to nicotinamide adenine dinucleotide (NADH) have been made on a case-by-case basis with varying degrees of success. Here we present a straightforward recipe for altering the cofactor specificity of a class of NADPH-dependent oxidoreductases, the ketol-acid reductoisomerases (KARIs). Combining previous results for an engineered NADH-dependent variant of Escherithia coil KARI with available KARI crystal structures and a comprehensive KARI-sequence alignment, we identified key cofactor specificity determinants and used this information to construct five KARIs with reversed cofactor preference. Additional directed evolution generated two enzymes having NADH-dependent catalytic efficiencies that are greater than the wild-type enzymes with NADPH. High-resolution structures of a wild-type/variant pair reveal the molecular basis of the cofactor switch. [ABSTRACT FROM AUTHOR]

Protein posttranslational modifications (PTMs), particularly phosphorylation, dramatically expand the complexity of cellular regulatory networks. Although cysteine (Cys) in various proteins can be subject to multiple PTMs, its phosphorylation was previously considered a rare PTM with almost no regulatory role assigned. We report here that phosphorylation occurs to a reactive cysteine residue conserved in the staphylococcal accessary regulator A (SarA)/MarR family global transcriptional regulator A (MgrA) family of proteins, and is mediated by the eukaryotic-like kinase-phosphatase pair Stkl-Stp1 in Staphylococcus aureus. Cys-phosphorylation is crucial in regulating virulence determinant production and bacterial resistance to vancomycin. Cell wall-targeting antibiotics, such as vancomycin and ceftriaxone, inhibit the kinase activity of Stk1 and lead to decreased Cys-phosphorylation of SarA and MgrA. An in vivo mouse model of infection established that the absence of stp1, which results in elevated protein Cys-phosphorylation, significantly reduces staphylococcal virulence. Our data indicate that Cys-phosphorylation is a unique PTM that can play crucial roles in bacterial signaling and regulation. [ABSTRACT FROM AUTHOR]

PA-824 is a promising new compound for the treatment of tuberculosis that is currently undergoing human trials. Like its progenitors metronidazole and CGI-17341, PA-824 is a prodrug of the nitroimidazole class, requiring bioreductive activation of an aromatic nitro group to exert an antitubercular effect. We have confirmed that resistance to PA-824 (a nitroimidazo-oxazine) and CGI-17341 (a nitroimidazo-oxazole) is most commonly mediated by loss of a specific glucose-6-phosphate dehydrogenase (FGD1) or its deazaflavin cofactor F420, which together provide electrons for the reductive activation of this class of molecules. Although FGD1 and F420 are necessary for sensitivity to these compounds, they are not sufficient and require additional accessory proteins that directly interact with the nitroimidazole. To understand more proximal events in the reductive activation of PA-824, we examined mutants that were wild-type for both FGD1 and F420 and found that, although these mutants had acquired high-level resistance to PA-824 (and another nitroimidazo-oxazine), they retained sensitivity to CGI-17341 (and a related nitroimidazo-oxazole). Micro-array-based comparative genome sequencing of these mutants identified lesions in Rv3547, a conserved hypothetical protein with no known function. Complementation with intact Rv3547 fully restored sensitivity to nitroimidazo-oxazines and restored the ability of Mtb to metabolize PA-824. These results suggest that the sensitivity of Mtb to PA-824 and related compounds is mediated by a protein that is highly specific for subtle structural variations in these bicyclic nitroimidazoles. [ABSTRACT FROM AUTHOR]