It's a Trap! Aldolase-Prescribed C4 Deoxyradiofluorination Affords Intracellular Trapping and the Tracing of Fructose Metabolism by PET.

Fructose metabolism has been implicated in various diseases, including metabolic disorders, neurodegenerative disorders, cardiac disorders, and cancer. However, the limited availability of a quantitative imaging radiotracer has hindered its exploration in pathology and diagnostic imaging. Methods: We adopted a molecular design strategy based on the catalytic mechanism of aldolase, a key enzyme in fructolysis. We successfully synthesized a radiodeoxyfluorinated fructose analog, [18F]4-fluoro-4-deoxyfructose ([18F]4-FDF), in high molar activity. Results: Through heavy isotope tracing by mass spectrometry, we demonstrated that C4-deoxyfluorination of fructose led to effective trapping as fluorodeoxysorbitol and fluorodeoxyfructose-1-phosphate in vitro, unlike C1- and C6-fluorinated analogs that resulted in fluorolactate accumulation. This observation was consistent in vivo, where [18F]6-fluoro-6-deoxyfructose displayed substantial bone uptake due to metabolic processing whereas [18F]4-FDF did not. Importantly, [18F]4-FDF exhibited low uptake in healthy brain and heart tissues, known for their high glycolytic activity and background levels of [18F]FDG uptake. [18F]4-FDF PET/CT allowed for sensitive mapping of neuro- and cardioinflammatory responses to systemic lipopolysaccharide administration. Conclusion: Our study highlights the significance of aldolase-guided C4 radiodeoxyfluorination of fructose in enabling effective radiotracer trapping, overcoming limitations of C1 and C6 radioanalogs toward a clinically viable tool for imaging fructolysis in highly glycolytic tissues.

A Prospective Randomized Controlled Trial Investigating Quadriceps Versus Hamstring Tendon Autograft in Anterior Cruciate Ligament Reconstruction.

BACKGROUND: Numerous graft options are available when undertaking anterior cruciate ligament (ACL) reconstruction (ACLR), although a lack of high-quality evidence exists comparing quadriceps (QT) and hamstring (HT) autografts. PURPOSE: To investigate patient outcomes in patients undergoing HT versus QT ACLR. STUDY DESIGN: Randomized controlled trial; Level of evidence, 1. METHODS: After recruitment and randomization, 112 patients (HT = 55; QT = 57) underwent ACLR. Patients were assessed pre- and postoperatively (6 weeks and 3, 6, 12, and 24 months), with a range of patient-reported outcome measures (PROMs), graft laxity (KT-1000 arthrometer; primary outcome variable), active knee flexion and extension range of motion (ROM), peak isokinetic knee extensor and flexor strength, and a 6-hop performance battery. Limb symmetry indices (LSIs) were calculated for strength and hop measures. Secondary procedures, ACL retears, and contralateral ACL tears were reported. RESULTS: All PROMs and knee ROM measures significantly improved (P < .0001), and no other group differences (P > .05) were observed-apart from the Anterior Cruciate Ligament Return to Sport after Injury (ACL-RSI) score, which was significantly better in the HT group at 3 (P = .008), 6 (P = .010), and 12 (P = .014) months. No significant changes were observed in side-to-side laxity from 6 to 24 months (P = .105), and no group differences were observed (P = .487) at 6 (HT mean, 1.2; QT mean, 1.3), 12 (HT mean, 1.1; QT mean, 1.3), and 24 (HT mean, 1.1; QT mean, 1.2) months. While the HT group demonstrated significantly greater (P < .05) quadriceps strength LSIs at 6 and 12 months, the QT group showed significantly greater (P < .05) hamstring strength LSIs at 6, 12, and 24 months. The HT group showed significantly greater (P < .05) LSIs for the single horizontal (6 months), lateral (6 and 12 months), and medial (6 months) hop tests for distance. Up until 24 months, 1 patient (QT at 22 months) had a retear, with 2 contralateral ACL tears (QT at 19 months; HT at 23 months). Secondary procedures included 5 in the HT group (manipulation under anesthesia, notch debridement, meniscal repair, and knee arthroscopy for scar tissue) and 6 in the QT group (notch debridement, meniscal repair, knee arthroscopy for scar tissue, tibial tubercle transfer, and osteochondral autologous transplantation). CONCLUSION: Apart from the ACL-RSI, the 2 autograft groups compared well for PROMs, knee ROM, and laxity. However, greater hamstring strength LSIs were observed for the QT cohort, with greater quadriceps strength (and hop test) LSIs in the HT cohort. The longer-term review will continue to evaluate return to sports and later-stage reinjury between the 2 graft constructs. REGISTRATION: ACTRN12618001520224p (Australian New Zealand Clinical Trials Registry).

pUdOs: Concise Plasmids for Bacterial and Mammalian Cells.

The plasmids from the Université d'Ottawa (pUdOs) are 28 small plasmids each comprising one of four origins of replication and one of seven selection markers, which together afford flexible use in Escherichia coli and several related gram-negative bacteria. The promoterless multicloning site is insulated from upstream spurious promoters by strong transcription terminators and contains type IIP or IIS restriction sites for conventional or Golden Gate cloning. pUdOs can be converted into efficient expression vectors through the insertion of a promoter at the user's discretion. For example, we demonstrate the utility of pUdOs as the backbone for an improved version of a Type III Secretion System reporter in Shigella. In addition, we derive a series of pUdO-based mammalian expression vectors, affording distinct levels of expression and transfection efficiency comparable to commonly used mammalian expression plasmids. Thus, pUdOs could advantageously replace traditional plasmids in a wide variety of cell types and applications.

Development of hydrogel-based composite scaffolds containing eggshell particles for bone regeneration applications.

This study describes the development and characterization of novel composite scaffolds, made of an alginate-chitosan hydrogel matrix containing eggshell (ES) particles, for bone tissue engineering applications. Scaffolds with ES particles, either untreated or treated with phosphoric acid to create a nanotextured particle surface, were compared to scaffolds without particles. Results indicate that the nanotexturing process exposed occluded ES proteins orthologous to those in human bone extracellular matrix. Scaffolds with ES or nanotextured ES (NTES) particles had a higher porosity (81 ± 4% and 89 ± 5%, respectively) than scaffolds without particles (59 ± 5%) (p = .002 and p < .001, respectively). Scaffolds with NTES particles had a larger median pore size (113 µm [interquartile range [IQ]: 88-140 µm]) than scaffolds with ES particles (94 µm [IQ: 75-112 µm]) and scaffolds without particles (99 µm [IQ: 74-135 µm]) (p < .001 and p = .011, respectively). The compressive modulus of the scaffolds with ES or NTES particles remained low (3.69 ± 0.70 and 3.14 ± 0.62 kPa, respectively), but these scaffolds were more resistant to deformation following maximum compression than those without particles. Finally, scaffolds with ES or NTES particles allowed better retention of human mesenchymal stem cells during seeding (53 ± 12% and 57 ± 8%, respectively, vs. 17 ± 5% for scaffolds without particles; p < .001 in both cases), as well as higher cell viability up to 21 days of culture (67 ± 17% and 61 ± 11%, respectively, vs. 15 ± 7% for scaffolds without particles; p < .001 in both cases). In addition, alkaline phosphatase (ALP) activity increased up to 558 ± 164% on day 21 in the scaffolds with ES particles, and up to 567 ± 217% on day 14 in the scaffolds with NTES particles (p = .006 and p = .002, respectively, relative to day 0). Overall, this study shows that the physicochemical properties of the alginate-chitosan hydrogel scaffolds with ES or NTES particles are similar to those of cancellous bone. In addition, scaffolds with particles supported early osteogenic differentiation and therefore represent a promising new bone substitute, especially for non-load bearing applications.

Core-multi-shell design: unlocking multimodal capabilities in lanthanide-based nanoparticles as upconverting, T2-weighted MRI and CT probes.

Multimodal bioimaging probes merging optical imaging, magnetic resonance imaging (MRI), and X-ray computed tomography (CT) capabilities have attracted considerable attention due to their potential biomedical applications. Lanthanide-based nanoparticles are promising candidates for multimodal imaging because of their optical, magnetic and X-ray attenuation properties. We prepared a set of hexagonal-phase (ß)-NaGdF4:Yb,Er/NaGdF4/NaDyF4 core/shell/shell nanoparticles (Dy-CSS NPs) and demonstrated their optical/T2-weighted MRI/CT multimodal capabilities. A known drawback of multimodal probes that merge the upconverting Er3+/Yb3+ ion pair with magnetic Dy3+ ions for T2-weighted MRI is the loss of upconversion (UC) emission due to Dy3+ poisoning. Particular attention was paid to controlled nanoparticle architectures with tuned inner shell thicknesses separating Dy3+ and Er3+/Yb3+ to shed light on the distance-dependent loss of UC due to Yb3+ → Dy3+ energy transfer. Based on the Er3+ UC spectra and the excited state lifetime of Yb3+, a 4 nm thick NaGdF4 inner shell did not only restore but enhanced the UC emission. We further investigated the effect of the outer NaDyF4 shell thickness on the particles' magnetic and CT performance. MRI T2 relaxivity measurements in vitro at a magnetic field of 7 T performed on citrate-capped Dy-CSS NPs revealed that NPs with the thickest outer shell thickness (4 nm) exhibited the highest r2 value, with a superior T2 contrast effect compared to commercial iron oxide and other Dy-based T2 contrast agents. In addition, the citrate-capped Dy-CSS NPs were demonstrated suitable for CT in in vitro imaging phantoms at X-ray energies of 110 keV, rendering them interesting alternatives to clinically used iodine-based agents that operate at lower energies.

Cell-Impermeable Inhibitors Confirm That Intracellular Human Transglutaminase 2 Is Responsible for the Transglutaminase-Associated Cancer Phenotype.

Transglutaminase 2 (TG2) is a multifunctional enzyme primarily responsible for crosslinking proteins. Ubiquitously expressed in humans, TG2 can act either as a transamidase by crosslinking two substrates through formation of an Nε(ɣ-glutaminyl)lysine bond or as an intracellular G-protein. These discrete roles are tightly regulated by both allosteric and environmental stimuli and are associated with dramatic changes in the conformation of the enzyme. The pleiotropic nature of TG2 and multi-faceted activities have resulted in TG2 being implicated in numerous disease pathologies including celiac disease, fibrosis, and cancer. Targeted TG2 therapies have not been selective for subcellular localization, such that currently no tools exist to selectively target extracellular over intracellular TG2. Herein, we have designed novel TG2-selective inhibitors that are not only highly potent and irreversible, but also cell impermeable, targeting only extracellular TG2. We have also further derivatized the scaffold to develop probes that are intrinsically fluorescent or bear an alkyne handle, which target both intra- and extracellular TG2, in order to facilitate cellular labelling and pull-down assays. The fluorescent probes were internalized and imaged in cellulo, and provide the first implicit experimental evidence that by comparison with their cell-impermeable analogues, it is specifically intracellular TG2, and presumably its G-protein activity, that contributes to transglutaminase-associated cancer progression.

Direct mapping of kidney function by DCE-MRI urography using a tetrazinanone organic radical contrast agent.

Chronic kidney disease (CKD) and acute kidney injury (AKI) are ongoing global health burdens. Glomerular filtration rate (GFR) is the gold standard measure of kidney function, with clinical estimates providing a global assessment of kidney health without spatial information of kidney- or region-specific dysfunction. The addition of dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) to the anatomical imaging already performed would yield a 'one-stop-shop' for renal assessment in cases of suspected AKI and CKD. Towards urography by DCE-MRI, we evaluated a class of nitrogen-centered organic radicals known as verdazyls, which are extremely stable even in highly reducing environments. A glucose-modified verdazyl, glucoverdazyl, provided contrast limited to kidney and bladder, affording functional kidney evaluation in mouse models of unilateral ureteral obstruction (UUO) and folic acid-induced nephropathy (FAN). Imaging outcomes correlated with histology and hematology assessing kidney dysfunction, and glucoverdazyl clearance rates were found to be a reliable surrogate measure of GFR.

Silver nanoparticles with exceptional near-infrared absorbance for photoenhanced antimicrobial applications.

In this work, we outline a simple method for synthesizing decahedral and triangular silver nanoparticles using light to tune particle shape and spectral characteristics. Notably, we were able to generate triangular silver nanoparticles with exceptional absorbance in the near-infrared (NIR) region, with high spectral overlap with the biological window, making them particularly promising for biological applications. We further demonstrate that under complementary LED illumination, these excitable plasmonic particles display exceptional antibacterial properties, several orders of magnitude more potent than similar particles under dark conditions or under illumination that does not match particle absorbance. This work demonstrates the powerful effects that LED lights can have on the antibacterial activity of AgNPs, providing an inexpensive and easily implemented route to unlocking the full potential of AgNPs in photobiological applications.

Kneeling tolerance following intramedullary nailing or plate fixation of uncomplicated distal tibia fractures.

INTRODUCTION: Distal tibia fractures often occur in younger, high demand patients, though the literature surrounding management remains contentious. This study sought to quantitatively determine differences in kneeling ability and self-reported knee function in patients managed with either intramedullary nailing (IMN) or open reduction internal fixation (ORIF) with compression plating following distal tibia fracture to assist in the preoperative consent process. HYPOTHESIS: There is no difference in kneeling tolerance following either tibial nailing or plate fixation of distal tibia fractures. MATERIAL AND METHODS: Retrospective sampling of public hospital data with outpatient prospective functional testing were completed. The primary outcome measure was the Kneeling Test (KT). Secondary outcome measures were The Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and the Forgotten Joint Score (FJS) for the knee joint. There were 28 patients recruited (18 IMN and 10 ORIF) with a mean age of 44years. Mean overall follow-up was 13.3months (range 8-25, SD 3.6). All fractures had completely healed without postoperative complication. RESULTS: The IMN affected limb had a significantly worse overall kneeling function than their non-affected limb (mean KT: 70.4 vs. 94.9 respectively, p<0.005) Additionally, the IMN group performed significantly worse when compared to the ORIF group (mean KT 70.4 vs. 92.5 respectively, p<0.005). No significant differences (p>0.05) in kneeling function existed for the ORIF group when comparing affected to non-affected limbs. Secondary outcome analysis showed significantly worse overall WOMAC and FJS in the IMN group compared to the ORIF group (mean WOMAC 19.3 vs. 6.9 respectively, p=0.040; mean FJS 38.3 vs. 75.9 respectively, p=0.005). DISCUSSION: The use of intramedullary nailing for the treatment of distal tibia fracture results in a mean reduction of 20% in kneeling tolerance in comparison to ORIF. The resulting kneeling tolerance is comparable to that of patients post-total knee arthroplasty. The present findings should assist in the consent process for patients with high kneeling demands in sportive, professional or cultural pastimes. LEVEL OF EVIDENCE: IV; retrospective cohort study with quantitative outcome measurement.

Armeniaspirol analogues with more potent Gram-positive antibiotic activity show enhanced inhibition of the ATP-dependent proteases ClpXP and ClpYQ.

Antibiotics with fundamentally new mechanisms of action such as the armeniaspirols, which target the ATP-dependent proteases ClpXP and ClpYQ, must be developed to combat antimicrobial resistance. While the mechanism of action of armeniaspirol against Gram-positive bacteria is understood, little is known about the structure-activity relationship for its antibiotic activity. Based on the preliminary data showing that modifications of armeniaspirol's N-methyl group increased antibiotic potency, we probed the structure-activity relationship of N-alkyl armeniaspirol derivatives. A series of focused derivatives were synthesized and evaluated for antibiotic activity against clinically relevant pathogens including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus. Replacement of the N-methyl with N-hexyl, various N-benzyl, and N-phenethyl substituents led to substantial increases in antibiotic activity and potency for inhibition of both ClpYQ and ClpXP. Docking studies identified binding models for ClpXP and ClpYQ that were consistent with the inhibition data. This work confirms the role of ClpXP and ClpYQ in the mechanism of action of armeniaspirol and provides important lead compounds for further antibiotic development.

Phytoglycogen Encapsulation of Lanthanide-Based Nanoparticles as an Optical Imaging Platform with Therapeutic Potential.

Lanthanide-based upconverting nanoparticles (UCNPs) are largely sought-after for biomedical applications ranging from bioimaging to therapy. A straightforward strategy is proposed here using the naturally sourced polymer phytoglycogen to coencapsulate UCNPs with hydrophobic photosensitizers as an optical imaging platform and light-induced therapeutic agents. The resulting multifunctional sub-micrometer-sized luminescent beads are shown to be cytocompatible as carrier materials, which encourages the assessment of their potential in biomedical applications. The loading of UCNPs of various elemental compositions enables multicolor hyperspectral imaging of the UCNP-loaded beads, endowing these materials with the potential to serve as luminescent tags for multiplexed imaging or simultaneous detection of different moieties under near-infrared (NIR) excitation. Coencapsulation of UCNPs and Rose Bengal opens the door for potential application of these microcarriers for collagen crosslinking. Alternatively, coloading UCNPs with Chlorin e6 enables NIR-light triggered generation of reactive oxygen species. Overall, the developed encapsulation methodology offers a straightforward and noncytotoxic strategy yielding water-dispersible UCNPs while preserving their bright and color-tunable upconversion emission that would allow them to fulfill their potential as multifunctional platforms for biomedical applications.

Kneeling difficulty is common following anterior cruciate ligament reconstruction with hamstring autograft and correlates with outcome measures.

INTRODUCTION: Patients frequently have discomfort or difficulty with kneeling following anterior cruciate ligament reconstruction (ACLR). This study aimed to report the prevalence of, and reasons for, kneeling difficulty after ACLR with a hamstring autograft; and to investigate the association between the degree of kneeling difficulty, presence of concurrent meniscal surgery, and clinical outcomes, including patient-reported outcome measures (PROMs) and functional tests. MATERIALS AND METHODS: A total of 104 patients undergoing ACLR with ipsilateral hamstring autograft were enrolled. Participants completed a kneeling difficulty questionnaire and other PROMs including the International Knee Documentation Committee (IKDC) questionnaire, the Knee Injury and Osteoarthritis Outcome Score (KOOS), the Cincinnati Knee Rating System (CKRS), the Lysholm Knee Scoring Scale (LKS), the Tegner Activity Scale (TAS), the 36-Item Short Form Health Survey (SF-36), and the Knee Outcome Survey (KOS). Patients were also assessed objectively via peak isokinetic knee extensor and flexor strength, range of movement (ROM), and functional hop tests. RESULTS: The prevalence of kneeling difficulty on the operated knee was 77% and 54% at 1 and 2 years after ACLR, respectively. Strong associations were observed between kneeling difficulty and PROMs, ranging from CKRS at 1 year of r = 0.403 (95% CI 0.228-0.553, p < 0.001) to KOS at 2 years of r = 0.724 (95% CI 0.618, 0.804, p < 0.001). No associations were observed with age, body mass index, or knee ROM measures. Weak-to-moderate associations were demonstrated with functional hop tests. The degree of kneeling difficulty did not differ with concurrent meniscal surgery. CONCLUSIONS: Kneeling difficulty occurs in as much as 77% of patients following ACLR with hamstring grafts at 1 year, and 54% at 2 years. This has a moderate-to-very strong association with patient-reported assessment of knee pain, symptoms, sport and recreation, and knee-related quality of life. There appears to be no association with patient age, BMI, time from injury to surgery, knee ROM, or concurrent meniscal surgery. LEVEL OF EVIDENCE: IV.

The kneeling test is a valid method of assessing kneeling tolerance.

PURPOSE: No quantitative test exists to reliably assess kneeling tolerance before and after surgery. The aim of this study was to validate a kneeling test, designed to quantify kneeling tolerance. METHOD: A total of 179 participants (324 knees) were enrolled into the study, including 124 asymptomatic knees, 98 with osteoarthritis (OA), 85 following total knee arthroplasty (TKA) and 17 following anterior cruciate ligament reconstruction (ACLR). Patients were asked to kneel on a custom-built platform on a soft, firm and hard surface, at both 90° and 110° of knee flexion. A kneeling score of 0-100 was established for 90° and 110° with 100 being a complete absence of pain or discomfort. A linear mixed model with random intercept was used to estimate differences between conditions (healthy, OA, ACLR and TKA). Pearson's correlation coefficient was used to test the strength of the association between the kneeling test and the forgotten joint score (FJS) and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score. Test-retest reliability was estimated by the intraclass correlation coefficient (ICC). RESULTS: The kneeling test demonstrated good discriminative validity with differences at 110° between asymptomatic knees and knees with OA (difference = - 42, p < 0.001), following ACLR (- 12, p = 0.013) and TKA (- 26, p < 0.001). Similar differences were observed at 90°. The kneeling test demonstrated construct validity, with a moderately strong correlation observed between the kneeling test and the FJS and the WOMAC at 90° (FJS 0.474 [95%CI: 0.357, 0.577], WOMAC - 0.503 [- 0.389, - 0.602]) and 110° (FJS 0.579 [95% CI: 0.479, 0.665], WOMAC - 0.648 [- 0.560, - 0.722]). The ICC for the kneeling test at 90° and 110° was 0.843 (95% CI: 0.745, 0.905) and 0.926 (95% CI: 0.877, 0.956), respectively. CONCLUSIONS: The kneeling test is a valid technique to quantitatively determine kneeling tolerance. It can aid in the assessment and modification of current surgical techniques to improve patient outcomes. LEVEL OF EVIDENCE: III.

KATP channel deficiency in mouse FDB causes an impairment of energy metabolism during fatigue.

The skeletal muscle ATP-sensitive K+ (KATP) channel is crucial in preventing fiber damage and contractile dysfunction, possibly by preventing damaging ATP depletion. The objective of this study was to investigate changes in energy metabolism during fatigue in wild-type and inwardly rectifying K+ channel (Kir6.2)-deficient (Kir6.2-/-) flexor digitorum brevis (FDB), a muscle that lacks functional KATP channels. Fatigue was elicited with one tetanic contraction every second. Decreases in ATP and total adenylate levels were significantly greater in wild-type than Kir6.2-/- FDB during the last 2 min of the fatigue period. Glycogen depletion was greater in Kir6.2-/- FDB for the first 60 s, but not by the end of the fatigue period, while there was no difference in glucose uptake. The total amount of glucosyl units entering glycolysis was the same in wild-type and Kir6.2-/- FDB. During the first 60 s, Kir6.2-/- FDB generated less lactate and more CO2; in the last 120 s, Kir6.2-/- FDB stopped generating CO2 and produced more lactate. The ATP generated during fatigue from phosphocreatine, glycolysis (lactate), and oxidative phosphorylation (CO2) was 3.3-fold greater in Kir6.2-/- than wild-type FDB. Because ATP and total adenylate were significantly less in Kir6.2-/- FDB, it is suggested that Kir6.2-/- FDB has a greater energy deficit, despite a greater ATP production, which is further supported by greater glucose uptake and lactate and CO2 production in Kir6.2-/- FDB during the recovery period. It is thus concluded that a lack of functional KATP channels results in an impairment of energy metabolism.