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The 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle from ammonia-oxidizing Thaumarchaeota is currently considered the most energy-efficient aerobic carbon fixation pathway. The Nitrosopumilus maritimus 4-hydroxybutyryl-CoA synthetase (ADP-forming; Nmar_0206) represents one of several enzymes from this cycle that exhibit increased efficiency over crenarchaeal counterparts. This enzyme reduces energy requirements on the cell, reflecting thaumarchaeal success in adapting to low-nutrient environments. Here we show the structure of Nmar_0206 from Nitrosopumilus maritimus SCM1, which reveals a highly conserved interdomain linker loop between the CoA-binding and ATP-grasp domains. Phylogenetic analysis suggests the widespread prevalence of this loop and highlights both its underrepresentation within the PDB and structural importance within the (ATP-forming) acyl-CoA synthetase (ACD) superfamily. This linker is shown to have a possible influence on conserved interface interactions between domains, thereby influencing homodimer stability. These results provide a structural basis for the energy efficiency of this key enzyme in the modified 3HP/4HB cycle of Thaumarchaeota.

Pyrone-2,4-dicarboxylic acid (PDC) is a valuable polymer precursor that can be derived from the microbial degradation of lignin. The key enzyme in the microbial production of PDC is 4-carboxy-2-hydroxymuconate-6-semialdehyde (CHMS) dehydrogenase, which acts on the substrate CHMS. We present the crystal structure of CHMS dehydrogenase (PmdC from Comamonas testosteroni) bound to the cofactor NADP, shedding light on its three-dimensional architecture, and revealing residues responsible for binding NADP. Using a combination of structural homology, molecular docking, and quantum chemistry calculations, we have predicted the binding site of CHMS. Key histidine residues in a conserved sequence are identified as crucial for binding the hydroxyl group of CHMS and facilitating dehydrogenation with NADP. Mutating these histidine residues results in a loss of enzyme activity, leading to a proposed model for the enzyme's mechanism. These findings are expected to help guide efforts in protein and metabolic engineering to enhance PDC yields in biological routes to polymer feedstock synthesis.

α-l-(3-2)-Threofuranosyl nucleic acid (TNA) pairs with itself, cross-pairs with DNA and RNA, and shows promise as a tool in synthetic genetics, diagnostics, and oligonucleotide therapeutics. We studied in vitro primer insertion and extension reactions catalyzed by human trans-lesion synthesis (TLS) DNA polymerase η (hPol η) opposite a TNA-modified template strand without and in combination with O4-alkyl thymine lesions. Across TNA-T (tT), hPol η inserted mostly dAMP and dGMP, dTMP and dCMP with lower efficiencies, followed by extension of the primer to a full-length product. hPol η inserted dAMP opposite O4-methyl and -ethyl analogs of tT, albeit with reduced efficiencies relative to tT. Crystal structures of ternary hPol η complexes with template tT and O4-methyl tT at the insertion and extension stages demonstrated that the shorter backbone and different connectivity of TNA compared to DNA (3 → 2 versus 5 → 3, respectively) result in local differences in sugar orientations, adjacent phosphate spacings, and directions of glycosidic bonds. The 3-OH of the primers terminal thymine was positioned at 3.4 Å on average from the α-phosphate of the incoming dNTP, consistent with insertion opposite and extension past the TNA residue by hPol η. Conversely, the crystal structure of a ternary hPol η·DNA·tTTP complex revealed that the primers terminal 3-OH was too distant from the tTTP α-phosphate, consistent with the inability of the polymerase to incorporate TNA. Overall, our study provides a better understanding of the tolerance of a TLS DNA polymerase vis-à-vis unnatural nucleotides in the template and as the incoming nucleoside triphosphate.

Our laboratory reported the chemical synthesis and stereochemical assignment of the recently discovered peptide antibiotic clovibactin. The current paper reports an improved, gram-scale synthesis of the amino acid building block Fmoc-(2R,3R)-3-hydroxyasparagine-OH that enables structure-activity relationship studies of clovibactin. An alanine scan reveals that residues Phe1, d-Leu2, Ser4, Leu7, and Leu8 are important for antibiotic activity. The side-chain amide group of the rare d-Hyn5 residue is not essential to activity and can be replaced with a methyl group with a moderate loss of activity. An acyclic clovibactin analogue reveals that the macrolactone ring is essential to antibiotic activity. The enantiomer of clovibactin is active, albeit somewhat less so than clovibactin. A conformationally constrained clovibactin analogue retains moderate antibiotic activity, while a backbone N-methylated analogue is almost completely inactive. X-ray crystallography of these two analogues reveals that the macrolactone ring adopts a crown-like conformation that binds anions.

The voltage-dependent anion channel (VDAC) is a crucial mitochondrial protein that facilitates ion and metabolite exchange between mitochondria and the cytosol. Initially characterized over three decades ago, the structure of VDAC-1 was resolved in 2008, revealing a novel β-barrel protein architecture. This study presents the first room-temperature crystal structure of mouse VDAC-1 (mVDAC-1), which is a significant step toward understanding the channel's gating mechanism. The new structure, obtained at a 3.3 Å resolution, demonstrates notable differences from the previously determined cryogenic structure, particularly in the loop regions, which may be critical for the transition between the 'open' and 'closed' states of VDAC-1. Comparative analysis of the root-mean-square deviation (R.M.S.D.) and B-factors between the cryogenic and room-temperature structures suggests that these conformational differences, although subtle, are important for VDAC's functional transitions. The application of electric field-stimulated X-ray crystallography (EF-X) is proposed as a future direction to resolve the 'closed' state of VDAC-1 by inducing voltage-driven conformational changes in order to elucidate the dynamic gating mechanism of VDAC-1. Our findings have profound implications for understanding the molecular basis of VDAC's role in mitochondrial function and its regulation under physiological conditions.

IHMCIF (github.com/ihmwg/IHMCIF) is a data information framework that supports archiving and disseminating macromolecular structures determined by integrative or hybrid modeling (IHM), and making them Findable, Accessible, Interoperable, and Reusable (FAIR). IHMCIF is an extension of the Protein Data Bank Exchange/macromolecular Crystallographic Information Framework (PDBx/mmCIF) that serves as the framework for the Protein Data Bank (PDB) to archive experimentally determined atomic structures of biological macromolecules and their complexes with one another and small molecule ligands (e.g., enzyme cofactors and drugs). IHMCIF serves as the foundational data standard for the PDB-Dev prototype system, developed for archiving and disseminating integrative structures. It utilizes a flexible data representation to describe integrative structures that span multiple spatiotemporal scales and structural states with definitions for restraints from a variety of experimental methods contributing to integrative structural biology. The IHMCIF extension was created with the benefit of considerable community input and recommendations gathered by the Worldwide Protein Data Bank (wwPDB) Task Force for Integrative or Hybrid Methods (wwpdb.org/task/hybrid). Herein, we describe the development of IHMCIF to support evolving methodologies and ongoing advancements in integrative structural biology. Ultimately, IHMCIF will facilitate the unification of PDB-Dev data and tools with the PDB archive so that integrative structures can be archived and disseminated through PDB.

K-Ras is the most commonly mutated oncogene in human cancer. The recently approved non-small cell lung cancer drugs sotorasib and adagrasib covalently capture an acquired cysteine in K-Ras-G12C mutation and lock it in a signaling-incompetent state. However, covalent inhibition of G12D, the most frequent K-Ras mutation particularly prevalent in pancreatic ductal adenocarcinoma, has remained elusive due to the lack of aspartate-targeting chemistry. Here we present a set of malolactone-based electrophiles that exploit ring strain to crosslink K-Ras-G12D at the mutant aspartate to form stable covalent complexes. Structural insights from X-ray crystallography and exploitation of the stereoelectronic requirements for attack of the electrophile allowed development of a substituted malolactone that resisted attack by aqueous buffer but rapidly crosslinked with the aspartate-12 of K-Ras in both GDP and GTP state. The GTP-state targeting allowed effective suppression of downstream signaling, and selective inhibition of K-Ras-G12D-driven cancer cell proliferation in vitro and xenograft growth in mice.

KAI2 receptors, classified as plant α/β hydrolase enzymes, are capable of perceiving smoke-derived butenolide signals and endogenous yet unidentified KAI2-ligands (KLs). While the number of functional KAI2 receptors varies among land plant species, rice has only one KAI2 gene. Rice, a significant crop and representative of grasses, relies on KAI2-mediated Arbuscular mycorrhiza (AM) symbioses to flourish in traditionally arid and nutrient-poor environments. This study presents the first crystal structure of an active rice (Oryza sativa, Os) KAI2 hydrolase receptor. Our structural and biochemical analyses uncover grass-unique pocket residues influencing ligand sensitivity and hydrolytic activity. Through structure-guided analysis, we identify a specific residue whose mutation enables the increase or decrease of ligand perception, catalytic activity, and signal transduction. Furthermore, we investigate OsKAI2-mediated signaling by examining its ability to form a complex with its binding partner, the F-box protein DWARF3 (D3) ubiquitin ligase and subsequent degradation of the target substrate OsSMAX1, demonstrating the significant role of hydrophobic interactions in the OsKAI2-D3 interface. This study provides new insights into the diverse and pivotal roles of the OsKAI2 signaling pathway in the plant kingdom, particularly in grasses.

Engineered reverse hairpin constructs containing a partial C-heptad repeat (CHR) sequence followed by a short loop and full-length N-heptad repeat (NHR) were previously shown to form trimers in solution and to be nanomolar inhibitors of HIV-1 Env mediated fusion. Their target is the in situ gp41 fusion intermediate, and they have similar potency to other previously reported NHR trimers. However, their design implies that the NHR is partially covered by CHR, which would be expected to limit potency. An exposed hydrophobic pocket in the folded structure may be sufficient to confer the observed potency, or they may exist in a partially unfolded state exposing full length NHR. Here we examined their structure by crystallography, CD and fluorescence, establishing that the proteins are folded hairpins both in crystal form and in solution. We examined unfolding in the milieu of the fusion reaction by conducting experiments in the presence of a membrane mimetic solvent and by engineering a disulfide bond into the structure to prevent partial unfolding. We further examined the role of the hydrophobic pocket, using a hairpin-small molecule adduct that occluded the pocket, as confirmed by X-ray footprinting. The results demonstrated that the NHR region nominally covered by CHR in the engineered constructs and the hydrophobic pocket region that is exposed by design were both essential for nanomolar potency and that interaction with membrane is likely to play a role in promoting the required inhibitor structure. The design concepts can be applied to other Class 1 viral fusion proteins.

In the folded state, biomolecules exchange between multiple conformational states crucial for their function. However, most structural models derived from experiments and computational predictions only encode a single state. To represent biomolecules accurately, we must move towards modeling and predicting structural ensembles. Information about structural ensembles exists within experimental data from X-ray crystallography and cryo-electron microscopy. Although new tools are available to detect conformational and compositional heterogeneity within these ensembles, the legacy PDB data structure does not robustly encapsulate this complexity. We propose modifications to the macromolecular crystallographic information file (mmCIF) to improve the representation and interrelation of conformational and compositional heterogeneity. These modifications will enable the capture of macromolecular ensembles in a human and machine-interpretable way, potentially catalyzing breakthroughs for ensemble-function predictions, analogous to the achievements of AlphaFold with single-structure prediction.

Natural photosystems couple light harvesting to charge separation using a 'special pair' of chlorophyll molecules that accepts excitation energy from the antenna and initiates an electron-transfer cascade. To investigate the photophysics of special pairs independently of the complexities of native photosynthetic proteins, and as a first step toward creating synthetic photosystems for new energy conversion technologies, we designed C2-symmetric proteins that hold two chlorophyll molecules in closely juxtaposed arrangements. X-ray crystallography confirmed that one designed protein binds two chlorophylls in the same orientation as native special pairs, whereas a second designed protein positions them in a previously unseen geometry. Spectroscopy revealed that the chlorophylls are excitonically coupled, and fluorescence lifetime imaging demonstrated energy transfer. The cryo-electron microscopy structure of a designed 24-chlorophyll octahedral nanocage with a special pair on each edge closely matched the design model. The results suggest that the de novo design of artificial photosynthetic systems is within reach of current computational methods.

Attosecond science requires a substantial rethinking of how to make measurements on very short timescales; how to acquire the necessary equipment, technology, and personnel; and how to build a set of laboratories for such experiments. This entails a rejuvenation of the author in many respects, in the laboratory itself, with regard to students and postdocs, and in generating funding for research. It also brings up questions of what it means to do attosecond science, and the discovery of the power of X-ray spectroscopy itself, which complements the short timescales addressed. The lessons learned, expressed in the meanderings of this autobiographical article, may be of benefit to others who try to reinvent themselves.

Schistosomiasis, caused by a parasitic blood fluke of the genus Schistosoma, is a global health problem for which new chemotherapeutic options are needed. We explored the scaffold of gallinamide A, a natural peptidic metabolite of marine cyanobacteria that has previously been shown to inhibit cathepsin L-type proteases. We screened a library of 19 synthetic gallinamide A analogs and identified nanomolar inhibitors of the cathepsin B-type protease SmCB1, which is a drug target for the treatment of schistosomiasis mansoni. Against cultured S. mansoni schistosomula and adult worms, many of the gallinamides generated a range of deleterious phenotypic responses. Imaging with a fluorescent-activity-based probe derived from gallinamide A demonstrated that SmCB1 is the primary target for gallinamides in the parasite. Furthermore, we solved the high-resolution crystal structures of SmCB1 in complex with gallinamide A and its two analogs and describe the acrylamide covalent warhead and binding mode in the active site. Quantum chemical calculations evaluated the contribution of individual positions in the peptidomimetic scaffold to the inhibition of the target and demonstrated the importance of the P1 and P2 positions. Our study introduces gallinamides as a powerful chemotype that can be exploited for the development of novel antischistosomal chemotherapeutics.

Activating signal co-integrator complex 1 (ASCC1) acts with ASCC-ALKBH3 complex in alkylation damage responses. ASCC1 uniquely combines two evolutionarily ancient domains: nucleotide-binding K-Homology (KH) (associated with regulating splicing, transcriptional, and translation) and two-histidine phosphodiesterase (PDE; associated with hydrolysis of cyclic nucleotide phosphate bonds). Germline mutations link loss of ASCC1 function to spinal muscular atrophy with congenital bone fractures 2 (SMABF2). Herein analysis of The Cancer Genome Atlas (TCGA) suggests ASCC1 RNA overexpression in certain tumors correlates with poor survival, Signatures 29 and 3 mutations, and genetic instability markers. We determined crystal structures of Alvinella pompejana (Ap) ASCC1 and Human (Hs) PDE domain revealing high-resolution details and features conserved over 500 million years of evolution. Extending our understanding of the KH domain Gly-X-X-Gly sequence motif, we define a novel structural Helix-Clasp-Helix (HCH) nucleotide binding motif and show ASCC1 sequence-specific binding to CGCG-containing RNA. The V-shaped PDE nucleotide binding channel has two His-Φ-Ser/Thr-Φ (HXT) motifs (Φ being hydrophobic) positioned to initiate cyclic phosphate bond hydrolysis. A conserved atypical active-site histidine torsion angle implies a novel PDE substrate. Flexible active site loop and arginine-rich domain linker appear regulatory. Small-angle X-ray scattering (SAXS) revealed aligned KH-PDE RNA binding sites with limited flexibility in solution. Quantitative evolutionary bioinformatic analyses of disease and cancer-associated mutations support implied functional roles for RNA binding, phosphodiesterase activity, and regulation. Collective results inform ASCC1's roles in transactivation and alkylation damage responses, its targeting by structure-based inhibitors, and how ASCC1 mutations may impact inherited disease and cancer.

Crystal structures of human long-chain acyl-CoA dehydrogenase (LCAD) and the catalytically inactive Glu291Gln mutant, have been determined. These structures suggest that LCAD harbors functions beyond its historically defined role in mitochondrial β-oxidation of long and medium-chain fatty acids. LCAD is a homotetramer containing one FAD per 43 kDa subunit with Glu291 as the catalytic base. The substrate binding cavity of LCAD reveals key differences which makes it specific for longer and branched chain substrates. The presence of Pro132 near the start of the E helix leads to helix unwinding that, together with adjacent smaller residues, permits binding of bulky substrates such as 3α, 7α, l2α-trihydroxy-5β-cholestan-26-oyl-CoA. This structural element is also utilized by ACAD11, a eucaryotic ACAD of unknown function, as well as bacterial ACADs known to metabolize sterol substrates. Sequence comparison suggests that ACAD10, another ACAD of unknown function, may also share this substrate specificity. These results suggest that LCAD, ACAD10, ACAD11 constitute a distinct class of eucaryotic acyl CoA dehydrogenases.

N-myristoyltransferase (NMT) is a promising antimalarial drug target. Despite biochemical similarities between Plasmodium vivax and human NMTs, our recent research demonstrated that high selectivity is achievable. Herein, we report PvNMT-inhibiting compounds aimed at identifying novel mechanisms of selectivity. Various functional groups are appended to a pyrazole moiety in the inhibitor to target a pocket formed beneath the peptide binding cleft. The inhibitor core group polarity, lipophilicity, and size are also varied to probe the water structure near a channel. Selectivity index values range from 0.8 to 125.3. Cocrystal structures of two selective compounds, determined at 1.97 and 2.43 Å, show that extensions bind the targeted pocket but with different stabilities. A bulky naphthalene moiety introduced into the core binds next to instead of displacing protein-bound waters, causing a shift in the inhibitor position and expanding the binding site. Our structure-activity data provide a conceptual foundation for guiding future inhibitor optimizations.

Imaging scaffolds composed of designed protein cages fused to designed ankyrin repeat proteins (DARPins) have enabled the structure determination of small proteins by cryogenic electron microscopy (cryo-EM). One particularly well characterized scaffold type is a symmetric tetrahedral assembly composed of 24 subunits, 12 A and 12 B, which has three cargo-binding DARPins positioned on each vertex. Here, the X-ray crystal structure of a representative tetrahedral scaffold in the apo state is reported at 3.8 Å resolution. The X-ray crystal structure complements recent cryo-EM findings on a closely related scaffold, while also suggesting potential utility for crystallographic investigations. As observed in this crystal structure, one of the three DARPins, which serve as modular adaptors for binding diverse `cargo proteins, present on each of the vertices is oriented towards a large solvent channel. The crystal lattice is unusually porous, suggesting that it may be possible to soak crystals of the scaffold with small (≤30 kDa) protein cargo ligands and subsequently determine cage-cargo structures via X-ray crystallography. The results suggest the possibility that cryo-EM scaffolds may be repurposed for structure determination by X-ray crystallography, thus extending the utility of electron-microscopy scaffold designs for alternative structural biology applications.

We combine X-ray-specific tracer particles, a photon counting detector, and a liquid metal jet anode X-ray source to achieve O(1 kHz) X-ray imaging speeds in the laboratory, 15× faster than previous comparable studies with O(50 μm) tracers. To examine the limits of this measurement technique we conduct three experiments: 2D and 3D X-ray particle velocimetry (XPV) of Poiseuille pipe flow, 3D XPV of flow around a Taylor bubble, and 3D scalar mixing with a laminar jet. These experiments demonstrate the performance improvement achievable by combining the aforementioned elements, the applicability to multiphase flows and deforming systems, and the potential to capture scalar and vector quantities simultaneously. Most importantly, these experiments are conducted with a laboratory-scale system, showing that in-lab X-ray particle velocimetry techniques are now becoming usable for a wider range of flows of interest. Furthermore, the design of XPV experiments is discussed to clarify the trade offs between achievable imaging speed, domain size and spatiotemporal resolution.

Macromolecular crystallography generally requires the recovery of missing phase information from diffraction data to reconstruct an electron-density map of the crystallized molecule. Most recent structures have been solved using molecular replacement as a phasing method, requiring an a priori structure that is closely related to the target protein to serve as a search model; when no such search model exists, molecular replacement is not possible. New advances in computational machine-learning methods, however, have resulted in major advances in protein structure predictions from sequence information. Methods that generate predicted structural models of sufficient accuracy provide a powerful approach to molecular replacement. Taking advantage of these advances, AlphaFold predictions were applied to enable structure determination of a bacterial protein of unknown function (UniProtKB Q63NT7, NCBI locus BPSS0212) based on diffraction data that had evaded phasing attempts using MIR and anomalous scattering methods. Using both X-ray and micro-electron (microED) diffraction data, it was possible to solve the structure of the main fragment of the protein using a predicted model of that domain as a starting point. The use of predicted structural models importantly expands the promise of electron diffraction, where structure determination relies critically on molecular replacement.

Enzymes populate ensembles of structures necessary for catalysis that are difficult to experimentally characterize. We use time-resolved mix-and-inject serial crystallography at an x-ray free electron laser to observe catalysis in a designed mutant isocyanide hydratase (ICH) enzyme that enhances sampling of important minor conformations. The active site exists in a mixture of conformations, and formation of the thioimidate intermediate selects for catalytically competent substates. The influence of cysteine ionization on the ICH ensemble is validated by determining structures of the enzyme at multiple pH values. Large molecular dynamics simulations in crystallo and time-resolved electron density maps show that Asp17 ionizes during catalysis and causes conformational changes that propagate across the dimer, permitting water to enter the active site for intermediate hydrolysis. ICH exhibits a tight coupling between ionization of active site residues and catalysis-activated protein motions, exemplifying a mechanism of electrostatic control of enzyme dynamics.

A wooden house frame consists of many different lumber pieces, but because of the regularity of these building blocks, the structure can be designed using straightforward geometrical principles. The design of multicomponent protein assemblies, in comparison, has been much more complex, largely owing to the irregular shapes of protein structures1. Here we describe extendable linear, curved and angled protein building blocks, as well as inter-block interactions, that conform to specified geometric standards; assemblies designed using these blocks inherit their extendability and regular interaction surfaces, enabling them to be expanded or contracted by varying the number of modules, and reinforced with secondary struts. Using X-ray crystallography and electron microscopy, we validate nanomaterial designs ranging from simple polygonal and circular oligomers that can be concentrically nested, up to large polyhedral nanocages and unbounded straight 'train track' assemblies with reconfigurable sizes and geometries that can be readily blueprinted. Because of the complexity of protein structures and sequence-structure relationships, it has not previously been possible to build up large protein assemblies by deliberate placement of protein backbones onto a blank three-dimensional canvas; the simplicity and geometric regularity of our design platform now enables construction of protein nanomaterials according to 'back of an envelope' architectural blueprints.

In our efforts to develop inhibitors selective for neuronal nitric oxide synthase (nNOS) over endothelial nitric oxide synthase (eNOS), we found that nNOS can undergo conformational changes in response to inhibitor binding that does not readily occur in eNOS. One change involves movement of a conserved tyrosine, which hydrogen bonds to one of the heme propionates, but in the presence of an inhibitor, changes conformation, enabling part of the inhibitor to hydrogen bond with the heme propionate. This movement does not occur as readily in eNOS and may account for the reason why these inhibitors bind more tightly to nNOS. A second structural change occurs upon the binding of a second inhibitor molecule to nNOS, displacing the pterin cofactor. Binding of this second site inhibitor requires structural changes at the dimer interface, which also occurs more readily in nNOS than in eNOS. Here, we used a combination of crystallography, mutagenesis, and computational methods to better understand the structural basis for these differences in NOS inhibitor binding. Computational results show that a conserved tyrosine near the primary inhibitor binding site is anchored more tightly in eNOS than in nNOS, allowing for less flexibility of this residue. We also find that the inefficiency of eNOS to bind a second inhibitor molecule is likely due to the tighter dimer interface in eNOS compared with nNOS. This study provides a better understanding of how subtle structural differences in NOS isoforms can result in substantial dynamic differences that can be exploited in the development of isoform-selective inhibitors.

Amyloid-β (Aβ) forms heterogeneous oligomers, which are implicated in the pathogenesis of Alzheimers disease (AD). Many Aβ oligomers consist of β-hairpin building blocks─Aβ peptides in β-hairpin conformations. β-Hairpins of Aβ can adopt a variety of alignments, but the role that β-hairpin alignment plays in the formation and heterogeneity of Aβ oligomers is poorly understood. To explore the effect of β-hairpin alignment on the oligomerization of Aβ peptides, we designed and studied two model peptides with two different β-hairpin alignments. Peptides Aβm17-36 and Aβm17-35 mimic two different β-hairpins that Aβ can form, the Aβ17-36 and Aβ17-35 β-hairpins, respectively. These hairpins are similar in composition but differ in hairpin alignment, altering the facial arrangements of the side chains of the residues that they contain. X-ray crystallography and SDS-PAGE demonstrate that the difference in facial arrangement between these peptides leads to distinct oligomer formation. In the crystal state, Aβm17-36 forms triangular trimers that further assemble to form hexamers, while Aβm17-35 forms tetrameric β-barrels. In SDS-PAGE, Aβm17-36 assembles to form a ladder of oligomers, while Aβm17-35 either assembles to form a dimer or does not assemble at all. The differences in the behavior of Aβm17-36 and Aβm17-35 suggest β-hairpin alignment as a source of the observed heterogeneity of Aβ oligomers.

Microtubule-associated protein tau (MAPT/tau) accumulates in a family of neurodegenerative diseases, including Alzheimer's disease (AD). In disease, tau is aberrantly modified by post-translational modifications (PTMs), including hyper-phosphorylation. However, it is often unclear which of these PTMs contribute to tau's accumulation or what mechanisms might be involved. To explore these questions, we focus on a cleaved proteoform of tau (tauC3), which selectively accumulates in AD and was recently shown to be degraded by its direct binding to the E3 ubiquitin ligase, CHIP. Here, we find that phosphorylation of tauC3 at a single residue, pS416, is sufficient to weaken its interaction with CHIP. A co-crystal structure of CHIP bound to the C-terminus of tauC3 reveals the mechanism of this clash, allowing design of a mutation (CHIPD134A) that partially restores binding and turnover of pS416 tauC3. We confirm that, in our models, pS416 is produced by the known AD-associated kinase, MARK2/Par-1b, providing a potential link to disease. In further support of this idea, an antibody against pS416 co-localizes with tauC3 in degenerative neurons within the hippocampus of AD patients. Together, these studies suggest a molecular mechanism for how phosphorylation at a discrete site contributes to accumulation of a tau proteoform.

Structure-based virtual screening is a key tool in early drug discovery, with growing interest in the screening of multi-billion chemical compound libraries. However, the success of virtual screening crucially depends on the accuracy of the binding pose and binding affinity predicted by computational docking. Here we develop a highly accurate structure-based virtual screen method, RosettaVS, for predicting docking poses and binding affinities. Our approach outperforms other state-of-the-art methods on a wide range of benchmarks, partially due to our ability to model receptor flexibility. We incorporate this into a new open-source artificial intelligence accelerated virtual screening platform for drug discovery. Using this platform, we screen multi-billion compound libraries against two unrelated targets, a ubiquitin ligase target KLHDC2 and the human voltage-gated sodium channel NaV1.7. For both targets, we discover hit compounds, including seven hits (14% hit rate) to KLHDC2 and four hits (44% hit rate) to NaV1.7, all with single digit micromolar binding affinities. Screening in both cases is completed in less than seven days. Finally, a high resolution X-ray crystallographic structure validates the predicted docking pose for the KLHDC2 ligand complex, demonstrating the effectiveness of our method in lead discovery.

Phytohormone levels are regulated through specialized enzymes, participating not only in their biosynthesis but also in post-signaling processes for signal inactivation and cue depletion. Arabidopsis thaliana (At) carboxylesterase 15 (CXE15) and carboxylesterase 20 (CXE20) have been shown to deplete strigolactones (SLs) that coordinate various growth and developmental processes and function as signaling molecules in the rhizosphere. Here, we elucidate the X-ray crystal structures of AtCXE15 (both apo and SL intermediate bound) and AtCXE20, revealing insights into the mechanisms of SL binding and catabolism. The N-terminal regions of CXE15 and CXE20 exhibit distinct secondary structures, with CXE15 characterized by an alpha helix and CXE20 by an alpha/beta fold. These structural differences play pivotal roles in regulating variable SL hydrolysis rates. Our findings, both in vitro and in planta, indicate that a transition of the N-terminal helix domain of CXE15 between open and closed forms facilitates robust SL hydrolysis. The results not only illuminate the distinctive process of phytohormone breakdown but also uncover a molecular architecture and mode of plasticity within a specific class of carboxylesterases.

The dysfunction of α and β cells in pancreatic islets can lead to diabetes. Many questions remain on the subcellular organization of islet cells during the progression of disease. Existing three-dimensional cellular mapping approaches face challenges such as time-intensive sample sectioning and subjective cellular identification. To address these challenges, we have developed a subcellular feature-based classification approach, which allows us to identify α and β cells and quantify their subcellular structural characteristics using soft X-ray tomography (SXT). We observed significant differences in whole-cell morphological and organelle statistics between the two cell types. Additionally, we characterize subtle biophysical differences between individual insulin and glucagon vesicles by analyzing vesicle size and molecular density distributions, which were not previously possible using other methods. These sub-vesicular parameters enable us to predict cell types systematically using supervised machine learning. We also visualize distinct vesicle and cell subtypes using Uniform Manifold Approximation and Projection (UMAP) embeddings, which provides us with an innovative approach to explore structural heterogeneity in islet cells. This methodology presents an innovative approach for tracking biologically meaningful heterogeneity in cells that can be applied to any cellular system.

Chitin is an abundant biopolymer and pathogen-associated molecular pattern that stimulates a host innate immune response. Mammals express chitin-binding and chitin-degrading proteins to remove chitin from the body. One of these proteins, Acidic Mammalian Chitinase (AMCase), is an enzyme known for its ability to function under acidic conditions in the stomach but is also active in tissues with more neutral pHs, such as the lung. Here, we used a combination of biochemical, structural, and computational modeling approaches to examine how the mouse homolog (mAMCase) can act in both acidic and neutral environments. We measured kinetic properties of mAMCase activity across a broad pH range, quantifying its unusual dual activity optima at pH 2 and 7. We also solved high-resolution crystal structures of mAMCase in complex with oligomeric GlcNAcn, the building block of chitin, where we identified extensive conformational ligand heterogeneity. Leveraging these data, we conducted molecular dynamics simulations that suggest how a key catalytic residue could be protonated via distinct mechanisms in each of the two environmental pH ranges. These results integrate structural, biochemical, and computational approaches to deliver a more complete understanding of the catalytic mechanism governing mAMCase activity at different pH. Engineering proteins with tunable pH optima may provide new opportunities to develop improved enzyme variants, including AMCase, for therapeutic purposes in chitin degradation.

In their folded state, biomolecules exchange between multiple conformational states that are crucial for their function. Traditional structural biology methods, such as X-ray crystallography and cryogenic electron microscopy (cryo-EM), produce density maps that are ensemble averages, reflecting molecules in various conformations. Yet, most models derived from these maps explicitly represent only a single conformation, overlooking the complexity of biomolecular structures. To accurately reflect the diversity of biomolecular forms, there is a pressing need to shift toward modeling structural ensembles that mirror the experimental data. However, the challenge of distinguishing signal from noise complicates manual efforts to create these models. In response, we introduce the latest enhancements to qFit, an automated computational strategy designed to incorporate protein conformational heterogeneity into models built into density maps. These algorithmic improvements in qFit are substantiated by superior Rfree and geometry metrics across a wide range of proteins. Importantly, unlike more complex multicopy ensemble models, the multiconformer models produced by qFit can be manually modified in most major model building software (e.g., Coot) and fit can be further improved by refinement using standard pipelines (e.g., Phenix, Refmac, Buster). By reducing the barrier of creating multiconformer models, qFit can foster the development of new hypotheses about the relationship between macromolecular conformational dynamics and function.

The receptor tyrosine kinase ROR2 mediates noncanonical WNT5A signaling to orchestrate tissue morphogenetic processes, and dysfunction of the pathway causes Robinow syndrome, brachydactyly B, and metastatic diseases. The domain(s) and mechanisms required for ROR2 function, however, remain unclear. We solved the crystal structure of the extracellular cysteine-rich (CRD) and Kringle (Kr) domains of ROR2 and found that, unlike other CRDs, the ROR2 CRD lacks the signature hydrophobic pocket that binds lipids/lipid-modified proteins, such as WNTs, suggesting a novel mechanism of ligand reception. Functionally, we showed that the ROR2 CRD, but not other domains, is required and minimally sufficient to promote WNT5A signaling, and Robinow mutations in the CRD and the adjacent Kr impair ROR2 secretion and function. Moreover, using function-activating and -perturbing antibodies against the Frizzled (FZ) family of WNT receptors, we demonstrate the involvement of FZ in WNT5A-ROR signaling. Thus, ROR2 acts via its CRD to potentiate the function of a receptor super-complex that includes FZ to transduce WNT5A signals.

SignificanceX-ray-induced acoustic computed tomography (XACT) offers a promising approach to biomedical imaging, leveraging X-ray absorption contrast. It overcomes the shortages of traditional X-ray, allowing for more advanced medical imaging.AimThe review focuses on the significance and draws onto the potential applications of XACT to demonstrate it as an innovative imaging technique.ApproachThis review navigates the expanding landscape of XACT imaging within the biomedical sphere. Integral topics addressed encompass the refinement of imaging systems and the advancement in image reconstruction algorithms. The review particularly emphasizes XACT's significant biomedical applications.ResultsKey uses, such as breast imaging, bone density maps for osteoporosis, and X-ray molecular imaging, are highlighted to demonstrate the capability of XACT. A unique niche for XACT imaging is its application in in vivo dosimetry during radiotherapy, which has been validated on patients.ConclusionsBecause of its unique property, XACT has great potential in biomedicine and non-destructive testing. We conclude by casting light on potential future avenues in this promising domain.

Background: Correctly diagnosing and accurately distinguishing mycoplasma pneumonia in children has consistently posed a challenge in clinical practice, as it can directly impact the prognosis of affected children. To address this issue, we analyzed chest X-rays (CXR) using various deep learning models to diagnose pediatric mycoplasma pneumonia. Methods: We collected 578 cases of children with mycoplasma infection and 191 cases of children with virus infection, with available CXR sets. Three deep convolutional neural networks (ResNet50, DenseNet121, and EfficientNetv2-S) were used to distinguish mycoplasma pneumonia from viral pneumonia based on CXR. Accuracy, area under the curve (AUC), sensitivity, and specificity were used to evaluate the performance of the model. Visualization was also achieved through the use of Class Activation Mapping (CAM), providing more transparent and interpretable classification results. Results: Of the three models evaluated, ResNet50 outperformed the others. Pretrained with the ZhangLabData dataset, the ResNet50 model achieved 80.00% accuracy in the validation set. The model also showed robustness in two test sets, with accuracy of 82.65 and 83.27%, and AUC values of 0.822 and 0.758. In the test results using ImageNet pre-training weights, the accuracy of the ResNet50 model in the validation set was 80.00%; the accuracy in the two test sets was 81.63 and 62.91%; and the corresponding AUC values were 0.851 and 0.776. The sensitivity values were 0.884 and 0.595, and the specificity values were 0.655 and 0.814. Conclusions: This study demonstrates that deep convolutional networks utilizing transfer learning are effective in detecting mycoplasma pneumonia based on chest X-rays (CXR). This suggests that, in the near future, such computer-aided detection approaches can be employed for the early screening of pneumonia pathogens. This has significant clinical implications for the rapid diagnosis and appropriate medical intervention of pneumonia, potentially enhancing the prognosis for affected children. [ABSTRACT FROM AUTHOR]

Recent breakthroughs in protein structure prediction have enhanced the precision and speed at which protein configurations can be determined. Additionally, molecular dynamics (MD) simulations serve as a crucial tool for capturing the conformational space of proteins, providing valuable insights into their structural fluctuations. However, the scope of MD simulations is often limited by the accessible timescales and the computational resources available, posing challenges to comprehensively exploring protein behaviors. Recently emerging approaches have focused on expanding the capability of AlphaFold2 (AF2) to predict conformational substates of protein. Here, we benchmark the performance of various workflows that have adapted AF2 for ensemble prediction and compare the obtained structures with ensembles obtained from MD simulations and NMR. We provide an overview of the levels of performance and accessible timescales that can currently be achieved with machine learning (ML) based ensemble generation. Significant minima of the free energy surfaces remain undetected. [Display omitted] • Ensemble prediction quality depends on training input to AlphaFold 2 (AF2) • MSA subsampling predicts ensembles but may miss key protein conformations • Current ensembles cannot reliably determine free energy, conformations, or properties • ⁠Ensemble data is crucial to improve conformational model accuracy Riccabona et al. underscore the importance of accurate structural data in predicting protein structural ensembles. They note that although rapid methods like MSA subsampling can generate ensembles, they often overlook functionally significant conformations, thereby missing crucial kinetic and thermodynamic insights. [ABSTRACT FROM AUTHOR]

The world first came to know the existence of COVID-19 (SARS-CoV-2) in December 2019. Initially, doctors struggled to diagnose the increasing number of patients due to less availability of testing kits. To help doctors primarily diagnose the virus, researchers around the world have come up with some radiology imaging techniques using the Convolutional Neural Network (CNN). Previously some research methods were based on X-ray images and others on CT scan images. Few research methods addressed both image types, with the proposed models limited to detecting only COVID and NORMAL cases. This limitation motivated us to propose a 42-layer CNN model that works for complex scenarios (COVID, NORMAL, and PNEUMONIA_VIRAL) and more complex scenarios (COVID, NORMAL, PNEUMONIA_VIRAL, and PNEUMONIA_BACTERIA). Furthermore, our proposed model indicates better performance than any other previously proposed models in the detection of COVID-19. [ABSTRACT FROM AUTHOR]

In this work, poly(4‐methyl‐1‐pentene)/polypropylene/poly(4‐methyl‐1‐pentene) tri‐layer films were prepared. The microstructure of the film was adjusted by annealing in the temperature range of 150–237.5°C. Afterward, the influence of annealing and stretching temperature on the cavitation behavior of the film was investigated by small‐angle x‐ray scattering (SAXS). Results showed that for the "butterfly" scattering in the early stage, the radius of gyration (Rg) and maximum dimension (Dmax) of voids were ca. 75 and 200 nm, which were hardly influenced by annealing. Whereas, the volume fraction of voids was enlarged obviously, suggesting that more voids were formed; for the "streak" scattering in the late stage, the length of voids (Lv) grew gradually with stretching, and the ultimate value of Lv was 432.5 nm for un‐annealed film and 176 nm for film annealed at 230°C. Simultaneously, the misorientation of voids was reduced from 0.33 to 0.18. As the stretching temperature was elevated from 30 to 45 and 60°C, Rg and Dmax maintained at 75 and 200 nm, and Lv was enlarged to 434 and 432 nm. At the same time, the number of voids was reduced obviously. As the stretching temperature was 90°C, the voids were suppressed. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the diagnostic accuracy of X-rays in detecting osteoarthritis (OA) in comparison to MRI. Study Design: Cross-sectional study. Setting: The Fauji Foundation Hospital, Rawalpindi. Period: August, 2023 to March, 2024. Methods: Total 350 patients consented to clinical interviews, physical examinations, standing radiographs, and MRIs. Standing, semi-flexed posteroanterior radiographs of the knees were obtained with proper alignment to accurately detect joint space narrowing. Manual measurements of joint space width were conducted by a blinded orthopedic surgeon using the Kellgren-Lawrence grading system. Data analysis was performed using SPSS 23, with frequencies, percentages, means, and standard deviations calculated for relevant variables. Diagnostic accuracy was assessed using a 2x2 table. Results: Of the 350 patients, 61.1% were up to 60 years old, and 38.9% were over 60 years old. Among those with positive MRI results, 70.1% also had a positive X-ray, while 29.6% did not. Cohen's Kappa was 0.377, indicating fair to moderate agreement. Sensitivity was 54.3%, specificity 82.4%, positive predictive value 70.1%, and negative predictive value 70.4%, with an overall diagnostic accuracy of 70.3%. Conclusion: X-rays demonstrated high specificity and moderate predictive values, but low sensitivity, suggesting that some OA cases may be missed if relying solely on X-rays. [ABSTRACT FROM AUTHOR]

The human Atg8 family member GABARAP is involved in numerous autophagy‐related and ‐unrelated processes. We recently observed that specifically the deficiency of GABARAP enhances epidermal growth factor receptor (EGFR) degradation upon ligand stimulation. Here, we report on two putative LC3‐interacting regions (LIRs) within EGFR, the first of which (LIR1) is selected as a GABARAP binding site in silico. Indeed, in vitro interaction studies reveal preferential binding of LIR1 to GABARAP and GABARAPL1. Our X‐ray data demonstrate interaction of core LIR1 residues FLPV with both hydrophobic pockets of GABARAP suggesting canonical binding. Although LIR1 occupies the LIR docking site, GABARAP Y49 and L50 appear dispensable in this case. Our data support the hypothesis that GABARAP affects the fate of EGFR at least in part through direct binding. [ABSTRACT FROM AUTHOR]

Background: A prominent anterior tibial tuberosity (or tibial tubercle) can be seen in ongoing Osgood–Schlatter disease (OSD) in teenagers or as a sequela of OSD in adults. Current radiological methods do not provide a true anatomical assessment of the tibial tuberosity; therefore, we proposed and developed a Magnetic Resonance Imaging (MRI)-based method for measuring the anterior tibial tuberosity index, aiming to deal with the current lack of effective techniques for accurately assessing these particular morphologic features. Methods: A retrospective study included 47 knees with tibial tuberosity measurements on both true sagittal MPR images of 3D proton density (PD)-weighted MRI sequences and lateral knee radiographs. The same landmarks were followed and the anterior tibial tuberosity index (ATTI) was measured. Results: The comparison of the results obtained by the two methods demonstrates that our method is reliable and reproducible with substantial inter- and intra-observer agreement. The intraclass correlation coefficient was 0.9250 (95% CI: 0.8654 to 0.9582), indicating excellent reliability between the two methods. A strong positive correlation was also identified, with a correlation coefficient of r = 0.8746 (95% CI: 0.7845 to 0.9286, p < 0.0001) between the two methods. No significant deviation from linearity was observed by analyzing the linear model validity using the cusum test (p = 0.62). Conclusions: Based on these results, we encourage the use of 3D PD-weighted MRI sequences for the measurement of the anterior tibial tuberosity on MRI in order to avoid unnecessary exposure to ionizing radiation and potentially obtain a more accurate measurement. Future larger studies should also explore the benefit of utilizing 3D sequences over 2D lateral projections to minimize measuring bias. [ABSTRACT FROM AUTHOR]

Objectives: Cervical spondylosis may lead to changes in the sagittal parameters of the neck and trigger compensatory alterations in systemic sagittal parameters. However, there is currently a dearth of comparative research on the changes and compensatory alterations to sagittal parameters resulting from different types of cervical spondylosis. This study compared the preoperative sagittal alignment sequences among patients with cervical spondylotic radiculopathy (CSR), ossification of the posterior longitudinal ligament (OPLL), and cervical spondylotic myelopathy (CSM) caused by factors resulting from non‐OPLL factors. Materials and Methods: Full length lateral X‐ray of the spine and cervical computed tomography (CT) of 256 patients (134 men, 122 women; mean age, 56.9 ± 9.5 years) were analyzed retrospectively. A total of 4096 radiomics features were measured through the lateral X‐ray by two spinal surgeons with extensive experience. The clinical symptoms measures were the Japanese Orthopaedic Association (JOA) score, number of hand actions in 10 s, hand‐grip strength, visual analog scale (VAS) score. Normally distributed data was compared using one‐way analysis of variance (ANOVA) for parametric variables and χ2 test were used to analyze the categorical data. Results: In the OPLL group, the C2‐C7 Cobb angle was greater than in the CSR and CSM groups (19.8 ± 10.4°, 13.3 ± 10.3°, and 13.9 ± 9.9°, respectively, p < 0.001). Additionally, the C7‐S1 SVA measure was found to be situated in the anterior portion with regards to the CSM and CSR groups (19.7 ± 58.4°, −6.3 ± 34.3° and −26.3 ± 32.9°, p < 0.001). Moreover, the number of individuals with C7‐S1 SVA >50 mm was significantly larger than the CSM group (26/69, 11/83, p < 0.001). In the CSR group, the TPA demonstrated smaller values compared to the OPLL group (8.8 ± 8.5°, 12.7 ± 10.2°, p < 0.001). Furthermore, the SSA was comparatively smaller as opposed to both the OPLL and CSM groups (49.6 ± 11.2°, 54.2 ± 10.8° and 54.3 ± 9.3°, p < 0.05). Conclusion: Patients with OPLL exhibit greater cervical lordosis than those with CSR and CSM. However, OPLL is more likely to result in spinal imbalance when compared to the CSM group. Furthermore, OPLL and CSM patients exhibit anterior trunk inclination and worse global spine sagittal parameters in comparison to CSR patients. [ABSTRACT FROM AUTHOR]

High-efficiency microcalorimeter arrays are required as imaging spectroscopy for X-ray microscopy in general and for astrophysics. We have developed in-house an array of Transition Edge Sensor (TES) X-ray microcalorimeters with mushroom-type absorbers which have a large area of 260 μ m square with multiple support stems. Insufficient thermal conductivity may degrade the energy resolution. We construct a 3D FEM simulation with thermal conduction and electric feedback circuit of TES. Estimated physical parameters were incorporated into the simulation to reproduce the measured pulse waveforms. As a result, the effect of the large absorber on the degradation of energy resolution was small for the mushroom-type absorber TES calorimeter. [ABSTRACT FROM AUTHOR]

Over 8% of women will be diagnosed with breast tumors (BT) in their lifetime. Tumors are formed by the uncontrollable development of tissues in a specific area of the body. They can be benign or malignant. The best survival rate can be expected with earlier screening and diagnosis of the tumor. To distinguish between benign and malignant tumors in x-ray images of the breast, segmentation of the tumor is a crucial first step. Screening mammography is an efficient method of detecting BT. As a result, the research presented two distinct deep learning models, termed SegNet and UNet architectures, to segment BT from mammograms. Datasets accessible to the public were utilized in the proposed system, specifically INbreast. Histogram equalization is used on datasets during preprocessing to improve the compressed areas and normalize the pixel dispersion. To avoid overfitting and boost the quantity of training data, augmentation techniques are employed. The metrics like the dice coefficient (DC) and the Intersection of Union (IoU) score are considered to evaluate the model. The metrics of the SegNets model are greater than the U-Net, as demonstrated by the experimental results. For the INbreast dataset, the SegNets achieve a maximum DC of 92.75% and an IoU score of 86.49%. [ABSTRACT FROM AUTHOR]

Hip migration percentage (MP) measured on anteroposterior pelvis radiographs is the gold standard to assess the severity of hip displacement in children with cerebral palsy (CP). Repeated exposure of these children to ionizing radiation under a hip surveillance program is undesirable. Recently, a semi-automatic approach to measure MP US on ultrasound (US) images was validated in a phantom study. This pilot in vivo study applied the previous phantom method and aimed to determine the reliability and accuracy of the MP US. Thirty-four children (23 boys and 11 girls) aged 8.9 ± 3.1 y old and diagnosed with CP were recruited. A total of 59 hips were scanned once, while 43 of these were scanned twice to evaluate the test-retest reliability. Two raters (R1 and R2) manually measured MP US ; procedures included selecting images of interest, cropping a region of interest and removing soft tissues on hip US images. Custom software was developed to measure MP automatically after the manual pre-image processing. The intra-class correlation coefficients (ICC 2,1) for the test-retest (R1), intra-rater (R1) and inter-rater (R1 vs R2) reliabilities were 0.90, 0.94 and 0.82, respectively. The standard error of measurement of MP US for all three evaluations was ≤3.0%. The mean absolute difference between MP US and MP X-ray and the percentage of MP US within clinical acceptance error of 10% for R1 and R2 were (R1: 6.2% ± 4.9%, 84.7%) and (R2: 7.6% ± 6.1%, 73.7%), respectively. This study demonstrated that US scans were repeatable and MP US could be measured reliably and accurately. [ABSTRACT FROM AUTHOR]

Chest X-ray, the established standard of confirming endotracheal tube (ETT) position, has important drawbacks including radiation exposure. Point-of-care airway ultrasound, which has been insufficiently studied in children, can overcome these problems. This was a prospective cross-sectional study done on children aged 2 months to 17 years undergoing intubation with cuffed ETT in the PICU. The ETT cuff was filled with saline and three ultrasonographic techniques were used– 1) Suprasternal (SS) method 2) Cricoid (CC) metho and 3) Tracheal ring (TR) method. Position of the ETT as determined by ultrasound and X-ray were compared. The main outcomes were sensitivity, specificity, and area under curve (AUC) for ultrasound-based methods vs. X-ray. For the TR method, concordance between the X-ray and ultrasound categories were taken. Total 62 patients were enrolled. The sensitivity and specificity of SS method were 71% (95% CI: 57–83%) and 100% (40–100%). The CC distance method had an AUC of 0.94 (95% CI: 0.86, 1.0). In the TR method, 98% of correct position on X-ray were correctly classified by USG. The agreement between X-ray and ultrasound categories with the cuff between the first and third tracheal rings, was very good [kappa (95% CI): 0.87 (0.70, 1.00), p ≤0.001)]. Bedside ultrasound is a good method to confirm ETT depth in children. The tracheal ring method had the best diagnostic accuracy and is easy to perform. The new method using cricoid cuff distance needs further validation in different ICU settings. • Chest X-ray the current gold standard for confirming correct endotracheal tube (ETT) position is unsafe. • Ultrasonography in comparison is safer, faster to obtain and easily accessible in ICUs. • We conducted this prospective cross-sectional study in 62 patients. • Three techniques of USG: suprasternal, cricoid and tracheal ring methods were studied. • We found USG to reliably confirm ET tube depth at the bedside. • Of the three methods, the tracheal ring method had the best diagnostic accuracy. [ABSTRACT FROM AUTHOR]

The development of portable X‐ray detectors is necessary for diagnosing fractures in unconscious patients in emergency situations. However, this is quite challenging because of the heavy weight of the scintillator and silicon photodetectors. The weight and thickness of X‐ray detectors can be reduced by replacing the silicon layer with an organic photodetectors. This study presents a novel bithienopyrroledione‐based polymer donor that exhibits excellent photodetection properties even in a thick photoactive layer (~700 nm), owing to the symmetric backbone and highly soluble molecular structure of bithienopyrroledione. The ability of bithienopyrroledione‐based polymer donor to strongly suppress the dark current density (Jd ~ 10−10 A cm−2) at a negative bias (−2.0 V) while maintaining high responsivity (R = 0.29 A W−1) even at a thickness of 700 nm results in a maximum shot‐noise‐limited specific detectivity of Dsh* = 2.18 × 1013 Jones in the organic photodetectors. Printed organic photodetectors are developed by slot‐die coating for use in X‐ray detectors, which exhibit Dsh* = 2.73 × 1012 Jones with clear rising (0.26 s) and falling (0.29 s) response times upon X‐ray irradiation. Detection reliability is also proven by linear response of the X‐ray detector, and the X‐ray detection limit is 3 mA. [ABSTRACT FROM AUTHOR]

If an unknown finite set C ⊂ Z 2 is cut by lines parallel to given directions, then one may count the number of points of C that are intercepted by each line, that is, the projections of C in the given directions. The inverse problem consists in reconstructing the set C , interpreted as a binary image, from the knowledge of its projections. In general, this challenging combinatorial problem, also related to the tomographic reconstruction of an unknown homogeneous object by means of X-rays, is ill-posed, meaning that different binary images exist that match the available projections. Therefore, as a preliminary step, one can try to find conditions to be imposed on the considered directions in order to limit the number of allowed solutions. In this paper we address the above problems for sets C contained in a finite assigned lattice grid, and generalize some results known in literature. First, we describe special sets of lattice directions, called simple cycles, and focus on some of their properties. Then we prove that uniqueness of reconstruction for binary images is guaranteed if and only if the line sums are computed along suitable simple cycles having even cardinality. As a second item, we prove that the unique binary solution can be explicitly reconstructed from a real-valued solution having minimal Euclidean norm. This leads to an explicit reconstruction algorithm, tested on four different phantoms and compared with previous results, which points out a significant improvement of the corresponding performance. [ABSTRACT FROM AUTHOR]

The anthracene‐appended Schiff base N1‐(anthracene‐9‐methylene)benzene‐1,2‐diamine and its Cu(II) complex has been synthesized and anchored on MCM‐41 support (MCM@CP@AnL@Cu 1). The synthesized catalyst was characterized by various analytical techniques including single crystal X‐ray. Malononitrile, 1,3‐dione, and aromatic aldehydes with various substituents were used in one‐pot, three‐component synthesis of 2‐amino‐3‐cyano‐4H‐pyran/chromene derivatives using above catalyst 1. All the products of the catalytic reaction were characterized by 1H and 13C NMR spectra. We have also proposed a probable reaction mechanism involved in the catalytic reaction. The present catalyst has several advantages like works in mild reaction conditions, that is, at 80 °C, gives higher yields up to 97%, has thermal stability up to 295 °C, and can be reused up to 5 cycles. The immobilized material did not show any considerable leaching or deterioration throughout the catalytic reactions, confirming that it possesses a practical benefit over homogeneous catalysis. The MCM@CP@AnL@Cu (1) is believed to be a good solid heterogeneous catalyst and meets the need of green chemistry for sustainable environment. To the best of our knowledge, this is first example of anthracene‐based Cu (II)‐Schiff base complexes immobilized on modified MCM‐41 surfaces for the multicomponent synthesis of 4H‐pyrans. [ABSTRACT FROM AUTHOR]

Gossypol and its derivatives arouse interest due to their broad spectrum of biological activities. Despite its wide potential application, there is no reported example of gossypol derivatives bearing stable radical functional groups. The first gossypol nitroxide hybrid compound was prepared here via formation of a Schiff base. By this approach, synthesis of a gossypol nitroxide conjugate was performed by condensation of gossypol with a 4-amino-TEMPO (4-amino-2,2,6,6-tetramethylpiperidin-1-oxyl) free radical, which afforded the target product in high yield. Its structure was proven by a combination of NMR and EPR spectroscopy, infrared spectroscopy, mass spectrometry, and high-resolution mass spectrometry. In addition, the structure of the gossypol nitroxide was determined by single-crystal X-ray diffraction measurements. In crystals, the paramagnetic Schiff base exists in an enamine–enamine tautomeric form. The tautomer is strongly stabilized by the intra- and intermolecular hydrogen bonds promoted by the resonance of π-electrons in the aromatic system. NMR analyses of the gossypol derivative proved that in solutions, the enamine–enamine tautomeric form prevailed. The gossypol nitroxide at micromolar concentrations suppressed the growth of tumor cells; however, compared to gossypol, the cytotoxicity of the obtained conjugate was substantially lower. [ABSTRACT FROM AUTHOR]

To design a comfortable hand support, the population-specific morphological characteristics of human hands must be known. Surface and bone anthropometric data from 177 radiograms of the left hands of an unselected sample of 87 subjects (training set) belonging to the Italian, female population (age 58 ± 12) were collected. Hand Length (HL) and Hand Width (HW) were respectively 172 ± 8 mm and 81 ± 4 mm; Hand Bone Length (HBL) was 179 ± 8 mm. A separate validation set consisting of 25 radiograms from 25 patients belonging to the same population was also collected. Bone and surface measurements were compared to validate the relationships proposed by Kong et al. Their approach applied to the validation set resulted in errors between 3.1% and 8.7%, which is almost as accurate (low bias) but somewhat less precise (higher mean errors) than what found by Kong et al. on their own sample belonging to the Korean population. Two methods were developed to predict the positions of the Metacarpo-phalangeal joints of the Index, Middle, and Ring fingers with reference to the Index-Middle interdigital crotch: method PM, based on the population mean, and method PR, which also considers the Hand Width. The overall average estimation error for method PM is 1.87 mm, while for method PR it is 1.69 mm. Method PR compared to method PM improves the precision by up to 32% in the x direction and 3% in the y direction. Based on these results, a 3D CAD hand model representing the target population was developed, which can be used for the design of population-specific ergonomic medical devices. [ABSTRACT FROM AUTHOR]

X-rays have a wide range of applications due to their ability to visualise the internal structure of bodies and objects. As well as diagnosing many medical conditions such as bones, internal organs and teeth, it is used in industry for quality control and materials testing, and for security checks in places such as airports and shopping centres. In addition, in the field of art, some artists offer innovative approaches with technological preferences such as X-rays for aesthetic expression. The use of X-rays in art is realised in two ways. Firstly, it is used for purposes such as dating by penetrating to the lower layers of artworks, determining the origin, originality and possible damage in the work, and determining the materials used. Secondly, X-rays are used directly for visual expression, allowing the final image to emerge. This research focuses on the use of Xrays in the field of art and is based on literature review on the subject. The aim of the research is to examine the role and impact of X-rays in the field of art and to analyse innovations in this field in the context of the relationship between art and technology. X-rays, and with them the curiosity about what lies behind the visible, allow for interaction between radiologists and artists working in different disciplines. The research findings indicate that among radiologists, there are those who use X-rays not only for medical diagnosis and treatment but also for various purposes such as taking artistic photographs. On the other hand, some artists use the technical characteristics of X-rays to focus on the aesthetic qualities, composition and tonal values of radiological images, as well as their symbolic, conceptual or emotional meaning. [ABSTRACT FROM AUTHOR]