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Background The diagnosis of periprosthetic joint infection (PJI) remains a serious clinical challenge. There is a pressing need for improved diagnostic testing methods; biomarkers offer one potentially promising approach. Questions/purposes We evaluated the diagnostic characteristics of 16 promising synovial fluid biomarkers for the diagnosis of PJI. Methods Synovial fluid was collected from 95 patients meeting the inclusion criteria of this prospective diagnostic study. All patients were being evaluated for a revision hip or knee arthroplasty, including patients with systemic inflammatory disease and those already receiving antibiotic treatment. The Musculoskeletal Infection Society (MSIS) definition was used to classify 29 PJIs and 66 aseptic joints. Synovial fluid samples were tested by immunoassay for 16 biomarkers optimized for use in synovial fluid. Sensitivity, specificity, and receiver operating characteristic curve analysis were performed to assess for diagnostic performance. Results Five biomarkers, including human α-defensin 1-3, neutrophil elastase 2, bactericidal/permeability-increasing protein, neutrophil gelatinase-associated lipocalin, and lactoferrin, correctly predicted the MSIS classification of all patients in this study, with 100% sensitivity and specificity for the diagnosis of PJI. An additional eight biomarkers demonstrated excellent diagnostic strength, with an area under the curve of greater than 0.9. Conclusions Synovial fluid biomarkers exhibit a high accuracy in diagnosing PJI, even when including patients with systemic inflammatory disease and those receiving antibiotic treatment. Considering that these biomarkers match the results of the more complex MSIS definition of PJI, we believe that synovial fluid biomarkers can be a valuable addition to the methods utilized for the diagnosis of infection. Level of Evidence Level II, diagnostic study. See Instructions for Authors for a complete description of levels of evidence.

Structure of a bile acid transporter Elevated cholesterol levels significantly increase the risk of atherosclerosis and cardiovascular diseases. Cholesterol is eliminated from the body following conversion to bile acids, so the apical sodium-dependent bile acid transporter (ASBT) that reabsorbs bile acid in the intestine is a major drug target for cholesterol-lowering therapy. The X-ray crystal structure of a bacterial homologue of ASBT bound to its bile acid substrate has now been determined. The substrate (taurocholate) is found in a large hydrophobic cavity between the protein's 'core' and 'panel' domains, suggesting a possible transport mechanism for this important biomolecule. High cholesterol levels greatly increase the risk of cardiovascular disease. About 50 per cent of cholesterol is eliminated from the body by its conversion into bile acids. However, bile acids released from the bile duct are constantly recycled, being reabsorbed in the intestine by the apical sodium-dependent bile acid transporter (ASBT, also known as SLC10A2). It has been shown in animal models that plasma cholesterol levels are considerably lowered by specific inhibitors of ASBT 1 , 2 , and ASBT is thus a target for hypercholesterolaemia drugs. Here we report the crystal structure of a bacterial homologue of ASBT from Neisseria meningitidis (ASBT NM ) at 2.2 Å. ASBT NM contains two inverted structural repeats of five transmembrane helices. A core domain of six helices harbours two sodium ions, and the remaining four helices pack in a row to form a flat, ‘panel’-like domain. Overall, the architecture of the protein is remarkably similar to the sodium/proton antiporter NhaA 3 , despite having no detectable sequence homology. The ASBT NM structure was captured with the substrate taurocholate present, bound between the core and panel domains in a large, inward-facing, hydrophobic cavity. Residues near this cavity have been shown to affect the binding of specific inhibitors of human ASBT 4 . The position of the taurocholate molecule, together with the molecular architecture, suggests the rudiments of a possible transport mechanism.

5‐hydroxymethylfurfural represents a key chemical in the drive towards a sustainable circular economy within the chemical industry. The final step in 5‐hydroxymethylfurfural production is the acid catalysed dehydration of fructose, for which supported organoacids are excellent potential catalyst candidates. Here we report a range of solid acid catalysis based on sulphonic acid grafted onto different porous silica nanosphere architectures, as confirmed by TEM, N2 porosimetry, XPS and ATR‐IR. All four catalysts display enhanced active site normalised activity and productivity, relative to alternative silica supported equivalent systems in the literature, with in‐pore diffusion of both substrate and product key to both performance and humin formation pathway. An increase in‐pore diffusion coefficient of 5‐hydroxymethylfurfural within wormlike and stellate structures results in optimal productivity. In contrast, poor diffusion within a raspberry‐like morphology decreases rates of 5‐hydroxymethylfurfural production and increases its consumption within humin formation. Sulphonic acid (RSO3H) functionalized porous silica nanospheres show high catalytic activity towards selective dehydration of fructose to 5‐hydroxymethylfurfural with pore architecture dictating performance and susceptibility to deactivation via humin by‐product formation. Catalyst performance is governed by in‐pore diffusion of substrate and product, which are assessed by diffusion NMR.

Anion exchanger 1 (AE1), also known as band 3 or SLC4A1, plays a key role in the removal of carbon dioxide from tissues by facilitating the exchange of chloride and bicarbonate across the plasma membrane of erythrocytes. An isoform of AE1 is also present in the kidney. Specific mutations in human AE1 cause several types of hereditary hemolytic anemias and/or distal renal tubular acidosis. Here we report the crystal structure of the band 3 anion exchanger domain (AE1CTD) at 3.5 angstroms. The structure is locked in an outward-facing open conformation by an inhibitor. Comparing this structure with a substrate-bound structure of the uracil transporter UraA in an inward-facing conformation allowed us to identify the anion-binding position in the AE1CTD, and to propose a possible transport mechanism that could explain why selected mutations lead to disease.

Three-dimensional cell culture has many advantages over monolayer cultures, and spheroids have been hailed as the best current representation of small avascular tumours in vitro. However their adoption in regular screening programs has been hindered by uneven culture growth, poor reproducibility and lack of high-throughput analysis methods for 3D. The objective of this study was to develop a method for a quick and reliable anticancer drug screen in 3D for tumour and human foetal brain tissue in order to investigate drug effectiveness and selective cytotoxic effects. Commercially available ultra-low attachment 96-well round-bottom plates were employed to culture spheroids in a rapid, reproducible manner amenable to automation. A set of three mechanistically different methods for spheroid health assessment (Spheroid volume, metabolic activity and acid phosphatase enzyme activity) were validated against cell numbers in healthy and drug-treated spheroids. An automated open-source ImageJ macro was developed to enable high-throughput volume measurements. Although spheroid volume determination was superior to the other assays, multiplexing it with resazurin reduction and phosphatase activity produced a richer picture of spheroid condition. The ability to distinguish between effects on malignant and the proliferating component of normal brain was tested using etoposide on UW228-3 medulloblastoma cell line and human neural stem cells. At levels below 10 µM etoposide exhibited higher toxicity towards proliferating stem cells, whereas at concentrations above 10 µM the tumour spheroids were affected to a greater extent. The high-throughput assay procedures use ready-made plates, open-source software and are compatible with standard plate readers, therefore offering high predictive power with substantial savings in time and money.

Short chain peptides are actively transported across membranes as an efficient route for dietary protein absorption and for maintaining cellular homeostasis. In mammals, peptide transport occurs via PepT1 and PepT2, which belong to the proton‐dependent oligopeptide transporter, or POT family. The recent crystal structure of a bacterial POT transporter confirmed that they belong to the major facilitator superfamily of secondary active transporters. Despite the functional characterization of POT family members in bacteria, fungi and mammals, a detailed model for peptide recognition and transport remains unavailable. In this study, we report the 3.3‐Å resolution crystal structure and functional characterization of a POT family transporter from the bacterium Streptococcus thermophilus . Crystallized in an inward open conformation the structure identifies a hinge‐like movement within the C‐terminal half of the transporter that facilitates opening of an intracellular gate controlling access to a central peptide‐binding site. Our associated functional data support a model for peptide transport that highlights the importance of salt bridge interactions in orchestrating alternating access within the POT family. Proton‐dependent oligopeptide transporters are required for the uptake of diet‐derived peptides in all kingdoms of life. The crystal structure of a bacterial transporter in the inward open conformation, together with a published structure in an occluded conformation, reveals the peptide transport mechanism.

The X-ray crystal structure of NapA, a Na + /H + antiporter from Thermus thermophilus , in an active, outward-facing state is reported; comparisons to the structure of a related transporter in a low pH/inactivated, inward-facing state show the conformational changes that occur when the membrane protein moves from an inward-facing to an outward-facing state, suggesting that Na + /H + antiporters operate by a two-domain rocking bundle model. Ins and outs of a Na + /H + antiporter This manuscript reports an X-ray crystal structure of NapA, a sodium/proton antiporter from Thermus thermophilus , in an active, outward-facing state. Antiporters of this type are active in the plasma membrane of all living cells, where they help to regulate intracellular pH, sodium concentration and cell volume. Comparison of this new structure to a previously published structure of a related transporter in a low pH/inactivated, inward-facing state reveals the conformational changes that occur when the membrane protein moves from an inward-facing to an outward-facing state. Sodium/proton (Na + /H + ) antiporters, located at the plasma membrane in every cell, are vital for cell homeostasis 1 . In humans, their dysfunction has been linked to diseases, such as hypertension, heart failure and epilepsy, and they are well-established drug targets 2 . The best understood model system for Na + /H + antiport is NhaA from Escherichia coli 1 , 3 , for which both electron microscopy and crystal structures are available 4 , 5 , 6 . NhaA is made up of two distinct domains: a core domain and a dimerization domain. In the NhaA crystal structure a cavity is located between the two domains, providing access to the ion-binding site from the inward-facing surface of the protein 1 , 4 . Like many Na + /H + antiporters, the activity of NhaA is regulated by pH, only becoming active above pH 6.5, at which point a conformational change is thought to occur 7 . The only reported NhaA crystal structure so far is of the low pH inactivated form 4 . Here we describe the active-state structure of a Na + /H + antiporter, NapA from Thermus thermophilus , at 3 Å resolution, solved from crystals grown at pH 7.8. In the NapA structure, the core and dimerization domains are in different positions to those seen in NhaA, and a negatively charged cavity has now opened to the outside. The extracellular cavity allows access to a strictly conserved aspartate residue thought to coordinate ion binding 1 , 8 , 9 directly, a role supported here by molecular dynamics simulations. To alternate access to this ion-binding site, however, requires a surprisingly large rotation of the core domain, some 20° against the dimerization interface. We conclude that despite their fast transport rates of up to 1,500 ions per second 3 , Na + /H + antiporters operate by a two-domain rocking bundle model, revealing themes relevant to secondary-active transporters in general.

Computational fluid dynamics (CFD) has aided the development, design, and analysis of hypersonic airbreathing propulsion technologies, such as scramjets. The complex flow field in a scramjet isolator has been the subject of intense interest and study for several decades. Many features of this flow field also occur in supersonic wind-tunnel nozzles and diffusers. Computational analysis of these topics has frequently provided immense insight into the actual functionality and performance. Research presented in this work supports scientific investigation and understanding of a less-researched topic, which is shock–shock interference and interaction with the boundary layer in supersonic internal flows, as well as the passive control of its adverse effects to prevent the onset of unstart in a scramjet isolator. This computational investigation is conducted on a backpressured isolator and a modified three-dimensional shock-tube to represent a scramjet isolator with ram effects provided by high-pressure gas and high-speed flow provided by a supersonic inflow. Computational results for the backpressured isolator have been validated against available measured time-averaged wall pressure data. The modified shock-tube provided an opportunity to study the shock–shock interference and shock–boundary-layer interaction effects that would occur in a scramjet isolator or a ram-accelerator when the high-speed flow from the inlet interacted with the shock produced due to the combustor pressure traveling and meeting in the isolator. An assessment of wall cooling effects on these phenomena is presented for both the backpressured isolator and the modified shock-tube.

Clathrin forms diverse lattice and cage structures that change size and shape rapidly in response to the needs of eukaryotic cells during clathrin-mediated endocytosis and intracellular trafficking. We present the cryo-EM structure and molecular model of assembled porcine clathrin, providing insights into interactions that stabilize key elements of the clathrin lattice, namely, between adjacent heavy chains, at the light chain–heavy chain interface and within the trimerization domain. Furthermore, we report cryo-EM maps for five different clathrin cage architectures. Fitting structural models to three of these maps shows that their assembly requires only a limited range of triskelion leg conformations, yet inherent flexibility is required to maintain contacts. Analysis of the protein–protein interfaces shows remarkable conservation of contact sites despite architectural variation. These data reveal a universal mode of clathrin assembly that allows variable cage architecture and adaptation of coated vesicle size and shape during clathrin-mediated vesicular trafficking or endocytosis.

The uric acid/xanthine H + symporter, UapA, is a high-affinity purine transporter from the filamentous fungus Aspergillus nidulans . Here we present the crystal structure of a genetically stabilized version of UapA (UapA-G411V Δ1–11 ) in complex with xanthine. UapA is formed from two domains, a core domain and a gate domain, similar to the previously solved uracil transporter UraA, which belongs to the same family. The structure shows UapA in an inward-facing conformation with xanthine bound to residues in the core domain. Unlike UraA, which was observed to be a monomer, UapA forms a dimer in the crystals with dimer interactions formed exclusively through the gate domain. Analysis of dominant negative mutants is consistent with dimerization playing a key role in transport. We postulate that UapA uses an elevator transport mechanism likely to be shared with other structurally homologous transporters including anion exchangers and prestin. UapA is a uric acid/xanthine H + symporter from a filamentous fungus. Here, the authors solve the crystal structure of the transporter in complex with xanthine revealing it to be a dimer, and this homodimerisation is proposed to be important for function.