Shuffled ATG8 interacting motifs form an ancestral bridge between UFMylation and autophagy.

UFMylation involves the covalent modification of substrate proteins with UFM1 (Ubiquitin-fold modifier 1) and is important for maintaining ER homeostasis. Stalled translation triggers the UFMylation of ER-bound ribosomes and activates C53-mediated autophagy to clear toxic polypeptides. C53 contains noncanonical shuffled ATG8-interacting motifs (sAIMs) that are essential for ATG8 interaction and autophagy initiation. However, the mechanistic basis of sAIM-mediated ATG8 interaction remains unknown. Here, we show that C53 and sAIMs are conserved across eukaryotes but secondarily lost in fungi and various algal lineages. Biochemical assays showed that the unicellular alga Chlamydomonas reinhardtii has a functional UFMylation pathway, refuting the assumption that UFMylation is linked to multicellularity. Comparative structural analyses revealed that both UFM1 and ATG8 bind sAIMs in C53, but in a distinct way. Conversion of sAIMs into canonical AIMs impaired binding of C53 to UFM1, while strengthening ATG8 binding. Increased ATG8 binding led to the autoactivation of the C53 pathway and sensitization of Arabidopsis thaliana to ER stress. Altogether, our findings reveal an ancestral role of sAIMs in UFMylation-dependent fine-tuning of C53-mediated autophagy activation.

Vacuolar Degradation of Plant Organelles.

Plants continuously remodel and degrade their organelles due to damage from their metabolic activities and environmental stressors, as well as an integral part of their cell differentiation programs. Whereas certain organelles use local hydrolytic enzymes for limited remodeling, most of pathways that control the partial or complete dismantling of organelles rely on vacuolar degradation. Specifically, selective autophagic pathways play a crucial role in recognizing and sorting plant organelle cargo for vacuolar clearance, especially under cellular stress conditions induced by factors like heat, drought, and damaging light. In these short reviews, we discuss the mechanisms that control the vacuolar degradation of chloroplasts, mitochondria, endoplasmic reticulum, Golgi, and peroxisomes, with an emphasis on autophagy, recently discovered selective autophagy receptors for plant organelles, and crosstalk with other catabolic pathways.

Molecular mechanisms of endomembrane trafficking in plants.

Endomembrane trafficking is essential for all eukaryotic cells. The best-characterized membrane trafficking organelles include the endoplasmic reticulum (ER), Golgi apparatus, early and recycling endosomes, multivesicular body, or late endosome, lysosome/vacuole, and plasma membrane. Although historically plants have given rise to cell biology, our understanding of membrane trafficking has mainly been shaped by the much more studied mammalian and yeast models. Whereas organelles and major protein families that regulate endomembrane trafficking are largely conserved across all eukaryotes, exciting variations are emerging from advances in plant cell biology research. In this review, we summarize the current state of knowledge on plant endomembrane trafficking, with a focus on four distinct trafficking pathways: ER-to-Golgi transport, endocytosis, trans-Golgi network-to-vacuole transport, and autophagy. We acknowledge the conservation and commonalities in the trafficking machinery across species, with emphasis on diversity and plant-specific features. Understanding the function of organelles and the trafficking machinery currently nonexistent in well-known model organisms will provide great opportunities to acquire new insights into the fundamental cellular process of membrane trafficking.

A blast fungus zinc-finger fold effector binds to a hydrophobic pocket in host Exo70 proteins to modulate immune recognition in rice.

Exocytosis plays an important role in plant-microbe interactions, in both pathogenesis and symbiosis. Exo70 proteins are integral components of the exocyst, an octameric complex that mediates tethering of vesicles to membranes in eukaryotes. Although plant Exo70s are known to be targeted by pathogen effectors, the underpinning molecular mechanisms and the impact of this interaction on infection are poorly understood. Here, we show the molecular basis of the association between the effector AVR-Pii of the blast fungus Maganaporthe oryzae and rice Exo70 alleles OsExo70F2 and OsExo70F3, which is sensed by the immune receptor pair Pii via an integrated RIN4/NOI domain. The crystal structure of AVR-Pii in complex with OsExo70F2 reveals that the effector binds to a conserved hydrophobic pocket in Exo70, defining an effector/target binding interface. Structure-guided and random mutagenesis validates the importance of AVR-Pii residues at the Exo70 binding interface to sustain protein association and disease resistance in rice when challenged with fungal strains expressing effector mutants. Furthermore, the structure of AVR-Pii defines a zinc-finger effector fold (ZiF) distinct from the MAX (Magnaporthe Avrs and ToxB-like) fold previously described for a majority of characterized M. oryzae effectors. Our data suggest that blast fungus ZiF effectors bind a conserved Exo70 interface to manipulate plant exocytosis and that these effectors are also baited by plant immune receptors, pointing to new opportunities for engineering disease resistance.

Circumvention of Topoisomerase IIα Intron 19 Intronic Polyadenylation in Acquired Etoposide-Resistant Human Leukemia K562 Cells.

DNA topoisomerase IIα (TOP2α; 170 kDa, TOP2α/170) is an essential enzyme for proper chromosome dysjunction by producing transient DNA double-stranded breaks and is an important target for DNA damage-stabilizing anticancer agents, such as etoposide. Therapeutic effects of TOP2α poisons can be limited due to acquired drug resistance. We previously demonstrated decreased TOP2α/170 levels in an etoposide-resistant human leukemia K562 subline, designated K/VP.5, accompanied by increased expression of a C-terminal truncated TOP2α isoform (90 kDa; TOP2α/90), which heterodimerized with TOP2α/170 and was a determinant of resistance by exhibiting dominant-negative effects against etoposide activity. Based on 3'-rapid amplification of cDNA ends, we confirmed TOP2α/90 as the translation product of a TOP2α mRNA in which a cryptic polyadenylation site (PAS) harbored in intron 19 (I19) was used. In this report, we investigated whether the resultant intronic polyadenylation (IPA) would be attenuated by blocking or mutating the I19 PAS, thereby circumventing acquired drug resistance. An antisense morpholino oligonucleotide was used to hybridize/block the PAS in TOP2α pre-mRNA in K/VP.5 cells, resulting in decreased TOP2α/90 mRNA/protein levels in K/VP.5 cells and partially circumventing drug resistance. Subsequently, CRISPR/CRISPR-associated protein 9 with homology-directed repair was used to mutate the cryptic I19 PAS (AATAAA→ACCCAA) to prevent IPA. Gene-edited clones exhibited increased TOP2α/170 and decreased TOP2α/90 mRNA/protein and demonstrated restored sensitivity to etoposide and other TOP2α-targeted drugs. Together, results indicated that blocking/mutating a cryptic I19 PAS in K/VP.5 cells reduced IPA and restored sensitivity to TOP2α-targeting drugs. SIGNIFICANCE STATEMENT: The results presented in this study indicate that CRISPR/CRISPR-associated protein 9 gene editing of a cryptic polyadenylation site (PAS) within I19 of the TOP2α gene results in the reversal of acquired resistance to etoposide and other TOP2-targeted drugs. An antisense morpholino oligonucleotide targeting the PAS also partially circumvented resistance.

Combined deletion of Mct8 and Dio2 impairs SVZ neurogliogenesis and olfactory function in adult mice.

Within the adult mouse subventricular zone (SVZ), neural stem cells (NSCs) produce neuroblasts and oligodendrocyte precursor cells (OPCs). T3, the active thyroid hormone, influences renewal and commitment of SVZ progenitors. However, how regulators of T3 availability affect these processes is less understood. Using Mct8/Dio2 knockout mice, we investigated the role of MCT8, a TH transporter, and DIO2, the T3-generating enzyme, in regulating adult SVZ-neurogliogenesis. Single-cell RNA-Seq revealed Mct8 expression in various SVZ cell types in WT mice, while Dio2 was enriched in neurons, astrocytes, and quiescent NSCs. The absence of both regulators in the knockout model dysregulated gene expression, increased the neuroblast/OPC ratio and hindered OPC differentiation. Immunostainings demonstrated compromised neuroblast migration reducing their supply to the olfactory bulbs, impairing interneuron differentiation and odor discrimination. These findings underscore the pivotal roles of MCT8 and DIO2 in neuro- and oligodendrogenesis, offering targets for therapeutic avenues in neurodegenerative and demyelinating diseases.

Bioinformatics Analysis Reveals E6 and E7 of HPV 16 Regulate Metabolic Reprogramming in Cervical Cancer, Head and Neck Cancer, and Colorectal Cancer through the PHD2-VHL-CUL2-ELOC-HIF-1α Axis.

Human papillomavirus 16 (HPV 16) infection is associated with several types of cancer, such as head and neck, cervical, anal, and penile cancer. Its oncogenic potential is due to the ability of the E6 and E7 oncoproteins to promote alterations associated with cell transformation. HPV 16 E6 and E7 oncoproteins increase metabolic reprogramming, one of the hallmarks of cancer, by increasing the stability of hypoxia-induced factor 1 α (HIF-1α) and consequently increasing the expression levels of their target genes. In this report, by bioinformatic analysis, we show the possible effect of HPV 16 oncoproteins E6 and E7 on metabolic reprogramming in cancer through the E6-E7-PHD2-VHL-CUL2-ELOC-HIF-1α axis. We proposed that E6 and E7 interact with VHL, CUL2, and ELOC in forming the E3 ubiquitin ligase complex that ubiquitinates HIF-1α for degradation via the proteasome. Based on the information found in the databases, it is proposed that E6 interacts with VHL by blocking its interaction with HIF-1α. On the other hand, E7 interacts with CUL2 and ELOC, preventing their binding to VHL and RBX1, respectively. Consequently, HIF-1α is stabilized and binds with HIF-1ß to form the active HIF1 complex that binds to hypoxia response elements (HREs), allowing the expression of genes related to energy metabolism. In addition, we suggest an effect of E6 and E7 at the level of PHD2, VHL, CUL2, and ELOC gene expression. Here, we propose some miRNAs targeting PHD2, VHL, CUL2, and ELOC mRNAs. The effect of E6 and E7 may be the non-hydroxylation and non-ubiquitination of HIF-1α, which may regulate metabolic processes involved in metabolic reprogramming in cancer upon stabilization, non-degradation, and translocation to the nucleus.

Use of CRISPR/Cas9 with Homology-Directed Repair to Gene-Edit Topoisomerase IIß in Human Leukemia K562 Cells: Generation of a Resistance Phenotype.

DNA topoisomerase IIß (TOP2ß/180; 180 kDa) is a nuclear enzyme that regulates DNA topology by generation of short-lived DNA double-strand breaks, primarily during transcription. TOP2ß/180 can be a target for DNA damage-stabilizing anticancer drugs, whose efficacy is often limited by chemoresistance. Our laboratory previously demonstrated reduced levels of TOP2ß/180 (and the paralog TOP2α/170) in an acquired etoposide-resistant human leukemia (K562) clonal cell line, K/VP.5, in part due to overexpression of microRNA-9-3p/5p impacting post-transcriptional events. To evaluate the effect on drug sensitivity upon reduction/elimination of TOP2ß/180, a premature stop codon was generated at the TOP2ß/180 gene exon 19/intron 19 boundary (AGAA//GTAA→ATAG//GTAA) in parental K562 cells (which contain four TOP2ß/180 alleles) by CRISPR/Cas9 editing with homology-directed repair to disrupt production of full-length TOP2ß/180. Gene-edited clones were identified and verified by quantitative polymerase chain reaction and Sanger sequencing, respectively. Characterization of TOP2ß/180 gene-edited clones, with one or all four TOP2ß/180 alleles mutated, revealed partial or complete loss of TOP2ß mRNA/protein, respectively. The loss of TOP2ß/180 protein correlated with decreased (2-{4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid)-induced DNA damage and partial resistance in growth inhibition assays. Partial resistance to mitoxantrone was also noted in the gene-edited clone with all four TOP2ß/180 alleles modified. No cross-resistance to etoposide or mAMSA was noted in the gene-edited clones. Results demonstrated the role of TOP2ß/180 in drug sensitivity/resistance in K562 cells and revealed differential paralog activity of TOP2-targeted agents. SIGNIFICANCE STATEMENT: Data indicated that CRISPR/Cas9 editing of the exon 19/intron 19 boundary in the TOP2ß/180 gene to introduce a premature stop codon resulted in partial to complete disruption of TOP2ß/180 expression in human leukemia (K562) cells depending on the number of edited alleles. Edited clones were partially resistant to mitoxantrone and XK469, while lacking resistance to etoposide and mAMSA. Results demonstrated the import of TOP2ß/180 in drug sensitivity/resistance in K562 cells and revealed differential paralog activity of TOP2-targeted agents.

A radiotherapy community data-driven approach to determine which complexity metrics best predict the impact of atypical TPS beam modeling on clinical dose calculation accuracy.

PURPOSE: To quantify the impact of treatment planning system beam model parameters, based on the actual spread in radiotherapy community data, on clinical treatment plans and determine which complexity metrics best describe the impact beam modeling errors have on dose accuracy. METHODS: Ten beam modeling parameters for a Varian accelerator were modified in RayStation to match radiotherapy community data at the 2.5, 25, 50, 75, and 97.5 percentile levels. These modifications were evaluated on 25 patient cases, including prostate, non-small cell lung, H&N, brain, and mesothelioma, generating 1,000 plan perturbations. Differences in the mean planned dose to clinical target volumes (CTV) and organs at risk (OAR) were evaluated with respect to the planned dose using the reference (50th-percentile) parameter values. Correlation between CTV dose differences, and 18 different complexity metrics were evaluated using linear regression; R-squared values were used to determine the best metric. RESULTS: Perturbations to MLC offset and transmission parameters demonstrated the greatest changes in dose: up to 5.7% in CTVs and 16.7% for OARs. More complex clinical plans showed greater dose perturbation with atypical beam model parameters. The mean MLC Gap and Tongue & Groove index (TGi) complexity metrics best described the impact of TPS beam modeling variations on clinical dose delivery across all anatomical sites; similar, though not identical, trends between complexity and dose perturbation were observed among all sites. CONCLUSION: Extreme values for MLC offset and MLC transmission beam modeling parameters were found to most substantially impact the dose distribution of clinical plans and careful attention should be given to these beam modeling parameters. The mean MLC Gap and TGi complexity metrics were best suited to identifying clinical plans most sensitive to beam modeling errors; this could help provide focus for clinical QA in identifying unacceptable plans.

Characterization of the Relative Change in Objective and Subjective Metrics by Baselining Patients Who Have Wearable Technology Before Total Knee Arthroplasty.

BACKGROUND: Wearable sensors and associated supporting technologies (ie, patient applications) can provide both objective (joint position, step counts, etc.) and subjective data (ie, pain scores and patient-reported outcome measures) to track a patient's episode of care. Establishing a subjective and objective baseline of a patient's experience may arguably be beneficial for multiple reasons, including setting recovery expectations for the patient and demonstrating the effectiveness or success of the intervention. METHODS: In this pilot study, we characterized a subset of patients (n = 82 from 7 surgeons) using a wearable sensor system at least 6 days before total knee arthroplasty and provided postsurgical data up to 50 days postintervention. The 5-day average before surgery for total step counts (activity), achieved flexion and extension on a progress test (functional limit) and visual analog scale daily pain score were calculated. The difference from baseline was then calculated for each patient for each day postsurgery and reported as averages. RESULTS: On average, a patient will experience a relative deficit of 4,000 steps immediately following surgery that will return to near-baseline levels 50 days postintervention. A 30° deficit in flexion and a 10° deficit in extension will return at a similar rate as steps. Relative pain scores will worsen with an increase of approximately 3 points immediately following surgery. However, pain will decrease by 2 points relative to baseline between 40 and 50 days. CONCLUSIONS: The results of this pilot study demonstrate a method to baseline a patient's presurgical subjective and objective data and to provide a reference for postsurgical recovery expectations. Applications for these data include benchmarking for evaluating intervention success as well as setting patient expectations.

Normative Values for Daily Functional Recovery Patterns Following Total Knee Arthroplasty.

BACKGROUND: Wearable devices provide the ability for clinical teams to continuously monitor patients' rehabilitation progress with objective data. Understanding expected recovery patterns following total knee arthroplasty (TKA) enables prompt identification of patients failing to meet these milestones. The aim of this study was to establish normative values for daily functional recovery in the first 6 weeks after TKA using a wearable device. METHODS: This prospective study included patients who underwent TKA between 2020 and 2023, treated by 11 surgeons from 8 institutions. Eligible participants were aged 18 or older, had a primary unilateral TKA, and owned a smartphone. Knee range of motion, total daily steps, cadence, and device usage were measured continuously over 6 weeks. Statistical analysis included analysis of variance using post hoc Tukey honest significant difference tests. RESULTS: The cohort of 566 participants had a mean age of 65 and 69 for men and women, respectively (range, 50 to 80). Women comprised 61% (n = 345) of study participants. There were 82% of women and 90% of men who had a body mass index > 30. The average daily wear time of the device was 12 hours (±4) for a total of 45 days (±27). Recovery was nonlinear, with the greatest gains in the first 3 weeks postsurgery for all metrics. Men demonstrated greater total daily step counts and cadence when compared to women. Obese patients demonstrated poorer performance when compared to lower body mass index patients. CONCLUSIONS: To our knowledge, this study presents the first normative data for tracking daily functional recovery in TKA patients using wearable sensors. Standardizing the TKA recovery timeline allows surgeons to isolate factors affecting patients' healing processes, accurately counsel them preoperatively, and intervene more promptly postoperatively when rehabilitation is not within standard recovery parameters.

Varying Complication Rates and Increased Costs in Technology-Assisted Total Hip Arthroplasty Versus Conventional Instrumentation in 1,372,300 Primary Total Hips.

BACKGROUND: The use of technology allows surgeons increased precision in component positioning in total hip arthroplasty (THA). The objective of this study was to compare (1) perioperative complications and (2) resource utilizations between robotic-assisted (RA) and computer-navigated (CN) versus conventional instrumenttaion (CI) THA. METHODS: A retrospective cohort study was performed using a large national database to identify patients undergoing unilateral, primary elective THA from January 1, 2016 to December 31, 2019 using RA, CN, or CI. There were 1,372,300 total patients identified and included RA (29,735), CN (28,480), and CI (1,314,085) THA. Demographics, complications, lengths of stay, dispositions, and costs were compared between the cohorts. Binary logistic regression analyses were performed. RESULTS: The use of RA THA led to lower rates of intraoperative fracture (0.22% versus 0.39%), delirium (0.1% versus 0.2%), postoperative anemia (14.4% versus 16.7%), higher myocardial infarction (0.13% versus 0.08%), renal failure (1.7% versus 1.6%), blood transfusion (2.0% versus 1.9%), and wound dehiscence (0.02% versus 0.01%) compared to CI THA. The use of CN led to lower rates of respiratory complication (0.5% versus 0.8%), renal failure (1.1% versus 1.6%), blood transfusion (1.3% versus 1.9%), and pulmonary embolism (0.02% versus 0.1%) compared to CI THA. Total costs were increased in RA ($17,729 versus $15,977) and CN ($22,529 versus $15,977). Lengths of hospital stay were decreased in RA (1.8 versus 1.9 days) and CN (1.7 versus 1.9 days). CONCLUSIONS: Perioperative complication rates vary in technology-assisted THA, with higher rates in RA THA and lower rates in CN THA, relative to CI THA. Both RA THA and CN THA were associated with more costs, shorter postoperative hospital stays, and higher rates of discharge home compared to CI THA.

Lower Perioperative Complication Rates and Shorter Lengths of Hospital Stay Associated With Technology-Assisted Total Knee Arthroplasty Versus Conventional Instrumentation in Primary Total Knee Arthroplasty.

BACKGROUND: The use of technology allows increased precision in component positioning in total knee arthroplasty (TKA). The objectives of this study were to compare (1) perioperative complications and (2) resource utilization between robotic-assisted (RA) and computer-navigated (CN) versus conventional (CI) TKA. METHODS: A retrospective cohort study was performed using a national database to identify patients undergoing unilateral, primary elective TKA from January 2016 to December 2019. A total of 2,174,685 patients were identified and included RA (69,445), CN (112,225), or CI (1,993,015) TKA. Demographics, complications, lengths of stay, dispositions, and costs were compared between the cohorts. Binary logistic regression analysis was performed. RESULTS: The RA TKA cohort had lower rates of intraoperative fracture (0.05 versus 0.08%, P < .05), respiratory complications (0.6 versus 1.1%, P < .05), renal failure (1.3 versus 1.7%, P < .05), delirium (0.1 versus 0.2%, P < .05), gastrointestinal complications (0.04 versus 0.09%, P < .05), postoperative anemia (8.9 versus 13.9%, P < .05), blood transfusion (0.4 versus 0.9%, P < .05), pulmonary embolism, and deep vein thrombosis (0.1 versus 0.2%, P < .05), and mortality (0.01 versus 0.02%, P < .05) compared to conventional TKA, though the cohort did have higher rates of myocardial infarction (0.09 versus 0.07%, P < .05). The CN cohort had lower rates of myocardial infarction (0.02 versus 0.07%, P < .05), respiratory complications (0.8 versus 1.1%, P < .05), renal failure (1.5 versus 1.7%, P < .05), blood transfusion (0.8 versus 0.9%, P < .05), pulmonary embolism (0.08 versus 0.2%, P < .05), and deep vein thrombosis (0.2 versus 0.2%, P < .05) over CI TKA. Total cost was increased in RA (16,190 versus $15,133, P < .05) and CN (17,448 versus $15,133, P < .05). However, the length of hospital stay was decreased in both RA (1.8 versus 2.2 days, P < .05) and CN (2.1 versus 2.2 days, P < .05). CONCLUSIONS: Technology-assisted TKA was associated with lower perioperative complication rates and faster recovery.

International Delphi Study on Wound Closure and Incision Management in Joint Arthroplasty Part 2: Total Hip Arthroplasty.

BACKGROUND: This modified Delphi study aimed to develop a consensus on optimal wound closure and incision management strategies for total hip arthroplasty (THA). Given the critical nature of wound care and incision management in influencing patient outcomes, this study sought to synthesize evidence-based best practices for wound care in THA procedures. METHODS: An international panel of 20 orthopedic surgeons from Europe, Canada, and the United States evaluated a targeted literature review of 18 statements (14 specific to THA and 4 related to both THA and total knee arthroplasty). There were 3 rounds of anonymous voting per topic using a modified 5-point Likert scale with a predetermined consensus threshold of ≥ 75% agreement necessary for a statement to be accepted. RESULTS: After 3 rounds of voting, consensus was achieved for all 18 statements. Notable recommendations for THA wound management included (1) the use of barbed sutures over non-barbed sutures (shorter closing times and overall cost savings); (2) the use of subcuticular sutures over skin staples (lower risk of superficial infections and higher patient preferences, but longer closing times); (3) the use of mesh-adhesives over silver-impregnated dressings (lower rate of wound complications); (4) for at-risk patients, the use of negative pressure wound therapy over other dressings (lower wound complications and reoperations, as well as fewer dressing changes); and (5) the use of triclosan-coated sutures (lower risk of surgical site infection) over standard sutures. CONCLUSIONS: Through a structured modified Delphi approach, a panel of 20 orthopedic surgeons reached consensus on all 18 statements pertaining to wound closure and incision management in THA. This study provides a foundational framework for establishing evidence-based best practices, aiming to reduce variability in patient outcomes and to enhance the overall quality of care in THA procedures.

Identifying Critical Evidence Gaps in Wound Closure and Incision Management After Total Knee Arthroplasty: Delphi Panel Insights.

INTRODUCTION: In orthopaedic surgery, particularly total knee arthroplasty (TKA), the management of surgical wounds is critical for optimal wound healing and successful patient outcomes. Despite advances in surgical techniques, challenges persist in effectively managing surgical wounds to prevent complications and infections. This study aimed to identify and address the critical evidence gaps in wound management in TKA, including preoperative optimization, intraoperative options, and for the avoidance of postoperative complications. These are important issues surrounding wound management, which is essential for improving patient recovery and the overall success of the surgery. METHODS: Utilizing the Delphi method, this study brought together 20 experienced orthopaedic surgeons from Europe and North America. Conducted from April to September 2023, the process involved three stages: an initial electronic survey, a virtual meeting, and a concluding electronic survey. The panel reviewed and reached a consensus on 26 specific statements about wound management in TKA based on a comprehensive literature review. During these three stages and after further panel review, an alternative goal of the Delphi panel was to also identify critical evidence gaps in the current understanding of wound management practices for TKA. RESULTS: While the panel reached consensus on various wound management practices, they highlighted several major evidence gaps. Also, there was general consensus on issues such as wound closure methods including the use of mesh-adhesive dressings, skin glue, staples, sutures (including barbed sutures),and negative-pressure wound therapy (NPWT). However, it was deemed necessary that further evidence needs to be generated to address the cost-effectiveness of each and develop best practices for promoting patient outcomes. The identification of these gaps points to areas requiring more in-depth research and improvements to enhance wound care in TKA. DISCUSSION: The identification of these major evidence gaps underscores the need for targeted research in wound management surrounding TKA. Addressing these evidence gaps is crucial for the future development of more effective, efficient, and patient-friendly wound care strategies. Future research should prioritize these areas, focusing on comparative effectiveness studies and further developing clear guidelines for the use of emerging technologies. Bridging these gaps has the potential to improve patient outcomes, reduce complications, and elevate the overall success rate of TKA surgeries.

International Delphi Study on Wound Closure and Dressing Management in Joint Arthroplasty: Part 1: Total Knee Arthroplasty.

BACKGROUND: The purpose of this modified Delphi study was to obtain consensus on wound closure and dressing management in total knee arthroplasty (TKA). METHODS: The Delphi panel included 20 orthopaedic surgeons from Europe and North America. There were 26 statements identified using a targeted literature review. Consensus was developed for the statements with up to three rounds of anonymous voting per topic. Panelists ranked their agreement with each statement on a five-point Likert scale. An a priori threshold of ≥ 75% was required for consensus. RESULTS: All 26 statements achieved consensus after three rounds of anonymous voting. Wound closure-related interventions that were recommended for use in TKA included: 1) closing in semi-flexion versus extension (superior range of motion); 2) using aspirin for venous thromboembolism prophylaxis over other agents (reduces wound complications); 3) barbed sutures over non-barbed sutures (lower wound complications, better cosmetic appearances, shorter closing times, and overall cost savings); 4) mesh-adhesives over other skin closure methods (lower wound complications, higher patient satisfaction scores, lower rates of readmission); 5) silver-impregnated dressings over standard dressings (lower wound complications, decreased infections, fewer dressing changes); 6) in high-risk patients, negative pressure wound therapy over other dressings (lower wound complications, decreased reoperations, fewer dressing changes); and 7) using triclosan-coated over non-antimicrobial-coated sutures (lower risks of surgical site infection). CONCLUSIONS: Using a modified Delphi approach, the panel achieved consensus on 26 statements pertaining to wound closure and dressing management in TKA. This study forms the basis for identifying critical evidence supported by clinical practice for wound management to help reduce variability, advance standardization, and ultimately improve outcomes during TKA. The results presented here can serve as the foundation for knowledge, education, and improved clinical outcomes for surgeons performing TKAs.

Biomechanical and histological comparison of two suture configurations for soft tissue grafts: speedtrap™ versus krackow stitch.

To compare 2 different graft preparation techniques to determine biomechanical strength and resultant tissue trauma evaluated by histology. Twelve common flexors of the finger's tendons were prepared with either tubulization (SpeedTrap™) or transtendon stiches (Orthocord™). The stiffness, resistance and energy at maximum load were tested for biomechanical assessment in both groups. After load testing, Samples were stained with hematoxylin and eosin (HE) to evaluate histological damage. We observe that the time to prepare tendons with SpeedTrap™ was 8.3 times faster (1:25 min) than traditional ones (15:02 min). In all cases, the mean values for SpeedTrap™ were higher in terms of strength, stiffness and energy at maximum load than for traditional suture but without significant difference (p > 0.05). The Krackow stitch produces greater structural damage to the collagen fibers while SpeedTrap™ maintains better organized arrangement of the fibers after tubulization preparation. With the results obtained, we can conclude that the tubulization technique allows faster graft preparation with less structural damage to the manipulated tissue without altering the biomechanical resistance provided by the transtendon suture technique.

HPLC-Based Metabolomic Analysis and Characterization of Amaranthus cruentus Leaf and Inflorescence Extracts for Their Antidiabetic and Antihypertensive Potential.

The aim of this study was to investigate the potential of Amaranthus cruentus flavonoids (quercetin, kaempferol, catechin, hesperetin, naringenin, hesperidin, and naringin), cinnamic acid derivatives (p-coumaric acid, ferulic acid, and caffeic acid), and benzoic acids (vanillic acid and 4-hydroxybenzoic acid) as antioxidants, antidiabetic, and antihypertensive agents. An analytical method for simultaneous quantification of flavonoids, cinnamic acid derivatives, and benzoic acids for metabolomic analysis of leaves and inflorescences from A. cruentus was developed with HPLC-UV-DAD. Evaluation of linearity, limit of detection, limit of quantitation, precision, and recovery was used to validate the analytical method developed. Maximum total flavonoids contents (5.2 mg/g of lyophilized material) and cinnamic acid derivatives contents (0.6 mg/g of lyophilized material) were found in leaves. Using UV-Vis spectrophotometry, the maximum total betacyanin contents (74.4 mg/g of lyophilized material) and betaxanthin contents (31 mg/g of lyophilized material) were found in inflorescences. The leaf extract showed the highest activity in removing DPPH radicals. In vitro antidiabetic activity of extracts was performed with pancreatic α-glucosidase and intestinal α-amylase, and compared to acarbose. Both extracts exhibited a reduction in enzyme activity from 57 to 74%. Furthermore, the in vivo tests on normoglycemic murine models showed improved glucose homeostasis after sucrose load, which was significantly different from the control. In vitro antihypertensive activity of extracts was performed with angiotensin-converting enzyme and contrasted to captopril; both extracts exhibited a reduction of enzyme activity from 53 to 58%. The leaf extract induced a 45% relaxation in an ex vivo aorta model. In the molecular docking analysis, isoamaranthin and isogomphrenin-I showed predictive binding affinity for α-glucosidases (human maltase-glucoamylase and human sucrase-isomaltase), while catechin displayed binding affinity for human angiotensin-converting enzyme. The data from this study highlights the potential of A. cruentus as a functional food.

Deformation Rate-Adaptive Conducting Polymers and Composites.

Materials are more easily damaged during accidents that involve rapid deformation. Here, a design strategy is described for electronic materials comprised of conducting polymers that defies this orthodox property, making their extensibility and toughness dynamically adaptive to deformation rates. This counterintuitive property is achieved through a morphology of interconnected nanoscopic core-shell micelles, where the chemical interactions are stronger within the shells than the cores. As a result, the interlinked shells retain material integrity under strain, while the rate of dissociation of the cores controls the extent of micelle elongation, which is a process that adapts to deformation rates. A prototype based on polyaniline shows a 7.5-fold increase in ultimate elongation and a 163-fold increase in toughness when deformed at increasing rates from 2.5 to 10 000% min-1 . This concept can be generalized to other conducting polymers and highly conductive composites to create "self-protective" soft electronic materials with enhanced durability under dynamic movement or deformation.

Effects of hsa-miR-9-3p and hsa-miR-9-5p on Topoisomerase IIß Expression in Human Leukemia K562 Cells with Acquired Resistance to Etoposide.

DNA topoisomerase IIα (TOP2α/170; 170 kDa) and topoisomerase IIß (TOP2ß/180; 180 kDa) are targets for a number of anticancer drugs, whose clinical efficacy is attenuated by chemoresistance. Our laboratory selected for an etoposide-resistant K562 clonal subline designated K/VP.5. These cells exhibited decreased TOP2α/170 and TOP2ß/180 expression. We previously demonstrated that a microRNA-9 (miR-9)-mediated posttranscriptional mechanism plays a role in drug resistance via reduced TOP2α/170 protein in K/VP.5 cells. Here, it is hypothesized that a similar miR-9 mechanism is responsible for decreased TOP2ß/180 levels in K/VP.5 cells. Both miR-9-3p and miR-9-5p are overexpressed in K/VP.5 compared with K562 cells, demonstrated by microRNA (miRNA) sequencing and quantitative polymerase chain reaction. The 3'-untranslated region (3'-UTR) of TOP2ß/180 contains miRNA recognition elements (MRE) for both miRNAs. Cotransfection of K562 cells with a luciferase reporter plasmid harboring TOP2ß/180 3'-UTR plus miR-9-3p or miR-9-5p mimics resulted in statistically significant decreased luciferase expression. miR-9-3p and miR-9-5p MRE mutations prevented this decrease, validating direct interaction between these miRNAs and TOP2ß/180 mRNA. Transfection of K562 cells with miR-9-3p/5p mimics led to decreased TOP2ß protein levels without a change in TOP2ß/180 mRNA and resulted in reduced TOP2ß-specific XK469-induced DNA damage. Conversely, K/VP.5 cells transfected with miR-9-3p/5p inhibitors led to increased TOP2ß/180 protein without a change in TOP2ß/180 mRNA and resulted in enhancement of XK469-induced DNA damage. Taken together, these results strongly suggest that TOP2ß/180 mRNA is translationally repressed by miR-9-3p/5p, that these miRNAs play a role in acquired resistance to etoposide, and that they are potential targets for circumvention of resistance to TOP2-targeted agents. SIGNIFICANCE STATEMENT: Results presented here indicate that miR-9-3p and miR-9-5p play a role in acquired resistance to etoposide via decreased DNA topoisomerase IIß 180 kDa protein levels. These findings contribute further information about and potential strategies for circumvention of drug resistance by modulation of microRNA levels. In addition, miR-9-3p and miR-9-5p overexpression in cancer chemoresistance may lead to future validation as biomarkers of responsiveness to DNA topoisomerase II-targeted therapy.