Kinetochores grip microtubules with directionally asymmetric strength.

For accurate mitosis, all chromosomes must achieve "biorientation," with replicated sister chromatids coupled via kinetochores to the plus ends of opposing microtubules. However, kinetochores first bind the sides of microtubules and subsequently find plus ends through a trial-and-error process; accurate biorientation depends on the selective release of erroneous attachments. Proposed mechanisms for error-correction have focused mainly on plus-end attachments. Whether erroneous side attachments are distinguished from correct side attachments is unknown. Here, we show that side-attached kinetochores are very sensitive to microtubule polarity, gripping sixfold more strongly when pulled toward plus versus minus ends. This directionally asymmetric grip is conserved in human and yeast subcomplexes, and it correlates with changes in the axial arrangement of subcomplexes within the kinetochore, suggesting that internal architecture dictates attachment strength. We propose that the kinetochore's directional grip promotes accuracy during early mitosis by stabilizing correct attachments even before both sisters have found plus ends.

Surgical Predictors of Clinical Outcome 6 Years After Revision ACL Reconstruction.

BACKGROUND: Revision anterior cruciate ligament (ACL) reconstruction has been documented to have inferior outcomes compared with primary ACL reconstruction. The reasons why remain unknown. PURPOSE: To determine whether surgical factors performed at the time of revision ACL reconstruction can influence a patient's outcome at 6-year follow-up. STUDY DESIGN: Cohort study; Level of evidence, 2. METHODS: Patients who underwent revision ACL reconstruction were identified and prospectively enrolled between 2006 and 2011. Data collected included baseline patient characteristics, surgical technique and pathology, and a series of validated patient-reported outcome instruments: Knee injury and Osteoarthritis Outcome Score (KOOS), International Knee Documentation Committee (IKDC) subjective form, Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), and Marx activity rating score. Patients were followed up for 6 years and asked to complete the identical set of outcome instruments. Regression analysis was used to control for baseline patient characteristics and surgical variables to assess the surgical risk factors for clinical outcomes 6 years after surgery. RESULTS: A total of 1234 patients were enrolled (716 men, 58%; median age, 26 years), and 6-year follow-up was obtained on 79% of patients (980/1234). Using an interference screw for femoral fixation compared with a cross-pin resulted in significantly better outcomes in 6-year IKDC scores (odds ratio [OR], 2.2; 95% CI, 1.2-3.9; P = .008) and KOOS sports/recreation and quality of life subscale scores (OR range, 2.2-2.7; 95% CI, 1.2-4.8; P < .01). Use of an interference screw compared with a cross-pin resulted in a 2.6 times less likely chance of having a subsequent surgery within 6 years. Use of an interference screw for tibial fixation compared with any combination of tibial fixation techniques resulted in significantly improved scores for IKDC (OR, 1.96; 95% CI, 1.3-2.9; P = .001); KOOS pain, activities of daily living, and sports/recreation subscales (OR range, 1.5-1.6; 95% CI, 1.0-2.4; P < .05); and WOMAC pain and activities of daily living subscales (OR range, 1.5-1.8; 95% CI, 1.0-2.7; P < .05). Use of a transtibial surgical approach compared with an anteromedial portal approach resulted in significantly improved KOOS pain and quality of life subscale scores at 6 years (OR, 1.5; 95% CI, 1.02-2.2; P≤ .04). CONCLUSION: There are surgical variables at the time of ACL revision that can modify clinical outcomes at 6 years. Opting for a transtibial surgical approach and choosing an interference screw for femoral and tibial fixation improved patients' odds of having a significantly better 6-year clinical outcome in this cohort.

Acoustic noise in a small-format 3.0-T neonatal MRI system.

BACKGROUND: Today, magnetic resonance imaging (MRI) is rarely used in managing the care of premature neonates. This is in large part due to the medical and logistical challenges associated with moving neonates from the neonatal intensive care unit (NICU) to the radiology department. Furthermore, acoustic noise associated with MR scanning poses safety concerns for both practitioners and neonatal patients. A small-format 3.0-T neonatal scanner was recently developed and placed within the NICU to address these logistical and acoustic challenges. OBJECTIVE: To compare acoustic noise measurements of a small-format 3.0-T neonatal MRI scanner with conventional adult-sized 1.5-T and 3.0-T MRI scanners using identical neonatal head imaging protocols. MATERIALS AND METHODS: Sound pressure level (SPL) measurements of a standard imaging protocol were made in a small-format neonatal 3.0-T MRI scanner as well as in adult-sized 1.5-T and 3.0-T scanners. SPL measurements were made with a Brüel & Kjær sound level meter model 2250. The statistical significance of the differences in SPL between scanners was determined using one-way ANOVA. RESULTS: Average sound pressure level values were measured in unweighted decibels (dB) and A-weighted decibels (dBA) for all imaging sequences in the protocol. The average A-weighted SPLs for the NICU from 1.5-T and 3.0-T MRI scanners were 81.02 ± 0.28 dBA, 87.00 ± 0.85 dBA, and 94.91 ± 0.65 dBA, respectively. SPLs at the isocenter of the NICU MRI scanner were 5.98 dBA quieter than in the 1.5-T scanner (P=0.007), and 13.89 dBA quieter than in the 3.0-T scanner (P<0.001). For staff standing next to the scanner, the NICU scanner was 20.24 dBA quieter than the 1.5-T scanner (P<0.001) and 19.28 dBA quieter than the 3.0-T scanner (P<0.001). CONCLUSION: The NICU 3.0-T MRI system is significantly quieter than conventional adult-sized MRI systems, improving safety for neonatal patients. Significant reductions in SPL were also noted inside the screen room where clinicians may be present during scanning.

PnictogenIII Dications Supported by BZIMPY Ligands.

Two homologous series of pnictogen(III) dications, stabilized by 2,6-bis(benzimidazole-2-yl)pyridine ligands have been prepared. Both series contain PnIII-X moieties (Pn = P, As, Sb, Bi; X = Cl or Ph) and have been fully characterized using spectroscopic methods including X-ray crystallography. The Lewis acidity of these compounds has also been probed by computational methods; the results suggest that the dictations are strong Lewis acids, with the PnCl2+ compounds being more acidic than the PnPh2+ compounds, and with Lewis acidity increasing from P to Bi, in both series. The PhP2+-containing compound was also found to be a versatile PIII transfer reagent, leading to new synthetic routes for various PhP-containing compounds. The redox chemistry of all compounds has also been probed using cyclic voltammetry and chemical reductions. In some cases the resulting PnI moieties could be trapped using diazabutadienes.

Radiomics approach for identifying radiation-induced normal tissue toxicity in the lung.

The rapidly evolving field of radiomics has shown that radiomic features are able to capture characteristics of both tumor and normal tissue that can be used to make accurate and clinically relevant predictions. In the present study we sought to determine if radiomic features can characterize the adverse effects caused by normal tissue injury as well as identify if human embryonic stem cell (hESC) derived extracellular vesicle (EV) treatment can resolve certain adverse complications. A cohort of 72 mice (n = 12 per treatment group) were exposed to X-ray radiation to the whole lung (3 × 8 Gy) or to the apex of the right lung (3 × 12 Gy), immediately followed by retro-orbital injection of EVs. Cone-Beam Computed Tomography images were acquired before and 2 weeks after treatment. In total, 851 radiomic features were extracted from the whole lungs and < 20 features were selected to train and validate a series of random forest classification models trained to predict radiation status, EV status and treatment group. It was found that all three classification models achieved significantly high prediction accuracies on a validation subset of the dataset (AUCs of 0.91, 0.86 and 0.80 respectively). In the locally irradiated lung, a significant difference between irradiated and unirradiated groups as well as an EV sparing effect were observed in several radiomic features that were not seen in the unirradiated lung (including wavelet-LLH Kurtosis, wavelet HLL Large Area High Gray Level Emphasis, and Gray Level Non-Uniformity). Additionally, a radiation difference was not observed in a secondary comparison cohort, but there was no impact of imaging machine parameters on the radiomic signature of unirradiated mice. Our data demonstrate that radiomics has the potential to identify radiation-induced lung injury and could be applied to predict therapeutic efficacy at early timepoints.

Investigating premotor corticospinal excitability in fast and slow voluntary contractions of the elbow flexors.

Corticospinal excitability (CSE) increases prior to a voluntary contraction; however, the relative contributions of premotor cortical and spinal mechanisms are poorly understood. It is unknown whether the intended voluntary contractile rate affects CSE. Eighteen young, healthy participants (nine females) completed isometric elbow flexion contractions targeting 50% maximal voluntary contraction (MVC) torque, at either fast (fast as possible) or slow (25% MVC/s) contractile rates. Participants were cued to contract with warning (red) and "GO" (green) visual signals. Magnetic and electric stimulations were applied to elicit motor evoked potentials (MEPs), cervicomedullary evoked potentials (CMEPs), and M-waves, in the surface electromyogram (EMG) recorded over the biceps brachii. MEPs and CMEPs were collected at 0, 25, 50 and 75% premotor reaction time (RT - defined as the time between the "GO" cue and onset of biceps brachii EMG) and compared to a resting baseline. MEP amplitude was greater than baseline at 75% RT (p=0.009), and CMEP amplitude was significantly increased at all RT points relative to baseline (p≤0.001). However, there were no differences in MEP and CMEP amplitudes when compared between fast and slow conditions (p≥0.097). Normalized to the CMEP, there was no difference in MEP amplitude from baseline in either contractile condition (p≥0.264). These results indicate that increased premotor CSE is a spinally-mediated response. Furthermore, premotor CSE is not influenced by the intended voluntary contractile rate. CMEP amplitudes were larger for females than males within the premotor RT period (p=0.038), demonstrating that premotor spinal excitability responses may be influenced by sex.

Retrospective Comparison of Quadratus Lumborum and Pericapsular Nerve Group Blocks for Postoperative Pain Management of Patients Undergoing Outpatient Hip Arthroscopy.

Introduction: Hip arthroscopy is commonly performed as an outpatient procedure and effective postoperative pain management is important to provide quality patient care and enable timely discharge. Multiple regional nerve blocks have been described for pain relief after hip arthroscopy, but there is no consensus on the optimal technique. This retrospective investigation aimed to compare quadratus lumborum (QL) and pericapsular nerve group (PENG) blocks to determine if there are differences in analgesic outcomes after outpatient hip arthroscopy. Methods: A total of 50 consecutive patients that received QL block and 50 that received PENG block for outpatient hip arthroscopy were identified and compared to determine if there were any differences in the primary outcome of total perioperative opioid consumption prior to discharge from the surgery center. Important secondary analgesic outcomes include postoperative opioid consumption, verbal rating scale (VRS) pain scores or total time in the recovery area. Summary statistics of relevant variables are compared and reported between study groups (QL versus PENG). Results: For QL and PENG groups, no significant differences were observed in total perioperative oral morphine equivalents (OME) (69.5 vs 60mg; p=0.40), postoperative OME (15 vs 15.3mg; p=0.96) or maximum pain scores in the recovery area (7.0 vs 6.0; p=0.41). Postoperatively, QL block patients were in PACU for a greater length of time after surgery than PENG block patients (89.5 vs 72 minutes; p<0.001). No patients had uncontrolled pain requiring emergency room visits or hospital admission within 24 hours. No neurologic complications or instances of motor weakness were reported after QL or PENG blocks. Conclusion: This retrospective study observed similar opioid requirements and pain scores for patients receiving QL versus PENG block for hip arthroscopy, though PENG block patients had shorter times in the recovery area. Prospective, controlled trials are required to further explore and confirm these findings.

Madarosis Among Breast Cancer Survivors.

BACKGROUND: Eyebrow and eyelash loss, known as madarosis, can occur after breast cancer-directed therapy. The purpose of this study was to ascertain the proportion of breast cancer survivors who experience madarosis, contributing factors, and associations between this symptom and quality of life. METHODS: Breast cancer survivors were invited to participate in an ongoing longitudinal cohort study as a part of the Mayo Clinic Breast Disease Registry (MCBDR). Consenting participants were mailed a survey approximately 1 year after diagnosis. The proportions of participants who reported eyebrow and eyelash loss were evaluated overall and according to treatment type. Quality of life (QOL) was also explored in this cohort. RESULTS: Eight hundred and thirty-eight breast cancer survivors responded to survey. The median age of survivors was 59.4 years (range 22-100 years), 315 (37%) had received chemotherapy (± endocrine therapy), 415 (50%) had received endocrine therapy only. Nearly half of participants reported eyebrow loss (49%) or eyelash loss (49%) that occurred after their diagnosis of breast cancer. Eyebrow loss was reported by 89% of chemotherapy recipients, by 27% of endocrine therapy only recipients, and by 19% of those not treated with either therapy. 102 (32%) of those with chemotherapy-associated eyebrow loss reported that it was complete. Eyelash loss was reported by 274 (87%) of chemotherapy recipients, 112 (27%) of endocrine therapy only recipients, and 23 (21%) of those who received neither therapy. CONCLUSIONS: Madarosis is a common symptom in breast cancer survivors and future investigation into the predictors and treatment of madarosis is needed.

The neuroendocrine transition in prostate cancer is dynamic and dependent on ASCL1.

Lineage plasticity is a hallmark of cancer progression that impacts therapy outcomes, yet the mechanisms mediating this process remain unclear. Here, we introduce a versatile in vivo platform to interrogate neuroendocrine lineage transformation throughout prostate cancer progression. Transplanted mouse prostate organoids with human-relevant driver mutations (Rb1-/-; Trp53-/-; cMyc+ or Pten-/-; Trp53-/-; cMyc+) develop adenocarcinomas, but only those with Rb1 deletion advance to aggressive, ASCL1+ neuroendocrine prostate cancer (NEPC) resistant to androgen receptor signaling inhibitors. Notably, this transition requires an in vivo microenvironment not replicated by conventional organoid culture. Using multiplexed immunofluorescence and spatial transcriptomics, we reveal that ASCL1+ cells arise from KRT8+ luminal cells, progressing into transcriptionally heterogeneous ASCL1+;KRT8- NEPC. Ascl1 loss in established NEPC causes transient regression followed by recurrence, but its deletion before transplantation abrogates lineage plasticity, resulting in castration-sensitive adenocarcinomas. This dynamic model highlights the importance of therapy timing and offers a platform to identify additional lineage plasticity drivers.

Nontuberculous Mycobacterial Pulmonary Disease: Patients, Principles, and Prospects.

Nontuberculous mycobacterial pulmonary disease (NTM-PD) is increasing in incidence globally and challenging to manage. The 2020 multisociety treatment guideline and the 2022 consensus recommendations provide comprehensive evidence-based guides to manage pulmonary diseases caused by the most common NTM. However, with >190 different NTM species that may require different multidrug regimens for treatment, the breadth and complexity of NTM-PD remain daunting for both patients and clinicians. In this narrative review, we aim to distill this broad, complex field into principles applicable to most NTM species and highlight important nuances, specifically elaborating on the presentation, diagnosis, principles of patient-centered care, principles of pathogen-directed therapy, and prospects of NTM-PD.

Inducing post-activation potentiation alters human motor unit behavior of the elbow extensors during unconstrained velocity contractions.

The purpose was to test whether inducing post-activation potentiation (PAP) altered motor unit (MU) activity during dynamic isotonic contractions. From 12 participants (3 females), 39 MUs were recorded from the anconeus (n=31) and lateral triceps brachii (n=8) with fine-wire electrodes during elbow extensions at 50 and 75% of peak power with, and without PAP. To induce PAP participants produced a 2s ramp conditioning contraction (CC) up to maximal isometric elbow extension with a 3s hold. Following the CC (~2s), independent electrical stimulation to the triceps and anconeus showed twitch torques were potentiated by 84 and 66%, respectively, (both p<0.001). Compared to baseline (i.e., without PAP), at both intensities (50 and 75%) PAP increased MU recruitment thresholds (40% and 80%, p<0.001) with lowered mean MU rates (-20 and -26%), and instantaneous rates at recruitment threshold (-26 and -25%) (all p<0.001). Firing rates increased 20% (p<0.001) from 50 to 75% power, but rates during potentiated contractions targeting 75% were lower than baseline at 50% (-10%, p<0.001). Dynamic contractions provide a more functional paradigm to assess MU activity with PAP and showed larger effects across a wider range of contractile intensities compared to previously described isometric tasks. Findings indicate that peripheral feedback from the potentiated muscle is likely not the primary mechanism in modifying MU behaviors as changes occurred at recruitment which is relatively insensitive to afferent feedback. Therefore, MU activity during dynamic contractions is responsive to activation history force potentiation and can make compensatory adjustments to optimize contractile output.

Arthroscopic Debridement and Fixation of Osteochondritis Dissecans Lesions of the Medial Femoral Condyle.

Unstable osteochondritis dissecans lesions of the medial femoral condyle have classically been treated with open reduction and fixation under direct visualization through an open arthrotomy. Given the value of avoiding open arthrotomies, we present an arthroscopic approach for lesion elevation, debridement, and fixation. The lesion is first elevated using an arthroscopic elevator, leaving a laterally based osseous hinge. Once elevated, fibrous debris is debrided from the base of the lesion. Subsequently, the fragment is reduced, and percutaneous transpatellar instrumentation is used for fixation. The use of this technique allows for excellent mobilization, debridement, and fixation of the osteochondritis dissecans lesion while minimizing violation of periarticular soft tissues.

Enzyme Activity Inhibition of α-Amylase Using Molecularly Imprinted Polymer (MIP) Hydrogel Microparticles.

Molecularly imprinted polymers (MIPs) are a class of synthetic recognition materials that offer a cost-effective and robust alternative to antibodies. While MIPs have found predominant use in biosensing and diagnostic applications, their potential for alternative uses, such as enzyme inhibition, remains unexplored. In this work, we synthesized a range of acrylamide-based hydrogel MIP microparticles (35 µm) specific for the recognition of α-amylase. These MIPs also showed good selectivity toward the target protein with over 96% binding of the target protein, compared with the control nonimprinted polymer (NIP) counterparts. Specificity of the MIPs was determined with the binding of nontarget proteins, trypsin, human serum albumin (HSA), and bovine serum albumin (BSA). The MIPs were further evaluated for their ability to inhibit α-amylase enzymatic activity, showing a significant decrease in activity. These findings highlight the potential of MIPs as enzyme inhibitors, suggesting an innovative application beyond their conventional use.

European Society of Intensive Care Medicine guidelines on end of life and palliative care in the intensive care unit.

The European Society of Intensive Care Medicine (ESICM) has developed evidence-based recommendations and expert opinions about end-of-life (EoL) and palliative care for critically ill adults to optimize patient-centered care, improving outcomes of relatives, and supporting intensive care unit (ICU) staff in delivering compassionate and effective EoL and palliative care. An international multi-disciplinary panel of clinical experts, a methodologist, and representatives of patients and families examined key domains, including variability across countries, decision-making, palliative-care integration, communication, family-centered care, and conflict management. Eight evidence-based recommendations (6 of low level of evidence and 2 of high level of evidence) and 19 expert opinions were presented. EoL legislation and the importance of respecting the autonomy and preferences of patients were given close attention. Differences in EoL care depending on country income and healthcare provision were considered. Structured EoL decision-making strategies are recommended to improve outcomes of patients and relatives, as well as staff satisfaction and mental health. Early integration of palliative care and the use of standardized tools for symptom assessment are suggested for patients at high risk of dying. Communication training for ICU staff and printed communication aids for families are advocated to improve outcomes and satisfaction. Methods for enhancing family-centeredness of care include structured family conferences and culturally sensitive interventions. Conflict-management protocols and strategies to prevent burnout among healthcare professionals are also considered. The work done to develop these guidelines highlights many areas requiring further research.

Accuracy and Reproducibility of Lipari-Szabo Order Parameters From Molecular Dynamics.

The Lipari-Szabo generalized order parameter probes the picosecond to nanosecond time scale motions of a protein and is useful for rationalizing a multitude of biological processes such as protein recognition and ligand binding. Although these fast motions are an important and intrinsic property of proteins, it remains unclear what simulation conditions are most suitable to reproduce methyl symmetry axis side chain order parameter data (Saxis2) from molecular dynamics simulations. In this study, we show that, while Saxis2 tends to converge within tens of nanoseconds, it is essential to run 10 to 20 replicas starting from configurations close to the experimental structure to obtain the best agreement with experimental Saxis2 values. Additionally, in a comparison of force fields, AMBER ff14SB outperforms CHARMM36m in accurately capturing these fast time scale motions, and we suggest that the origin of this performance gap is likely attributed to differences in side chain torsional parametrization and not due to differences in the global protein conformations sampled by the force fields. This study provides insight into obtaining accurate and reproducible Saxis2 values from molecular simulations and underscores the necessity of using replica simulations to compute equilibrium properties.

Essential tremor with tau pathology features seeds indistinguishable in conformation from Alzheimer's disease and primary age-related tauopathy.

Neurodegenerative tauopathies are characterized by the deposition of distinct fibrillar tau assemblies whose rigid core structures correlate with defined neuropathological phenotypes. Essential tremor (ET) is a progressive neurological disease that, in some cases, is associated with cognitive impairment and tau accumulation. Consequently, we explored the tau assembly conformation in ET patients with tau pathology using cytometry-based tau biosensor assays. These assays quantify tau prion seeding activity present in brain homogenates based on conversion of intracellular tau-fluorescent protein fusions from a soluble to an aggregated state. Prions exhibit seeding barriers, whereby a specific assembly structure cannot serve as a template for a native monomer if the amino acids are not compatible. We recently exploited the tau prion species barrier to define tauopathies by systematically substituting alanine (Ala) in the tau monomer and measuring its incorporation into seeded aggregates within biosensor cells. The Ala scan precisely classified the conformation of tau seeds from diverse tauopathies. We next studied 18 ET patient brains with tau pathology. Only one case had concurrent high amyloid-ß plaque pathology consistent with Alzheimer's disease (AD). We detected robust tau seeding activity in 9 (50%) of the patients. This predominantly localized to the temporal pole and temporal cortex. We examined 8 ET cases with the Ala scan and determined that the amino acid requirements for tau monomer incorporation into aggregates seeded from these ET brain homogenates were identical to those of AD and primary age-related tauopathy (PART), and completely distinct from other tauopathies such as corticobasal degeneration, chronic traumatic encephalopathy, and progressive supranuclear palsy. Based on these studies, tau assembly cores in a pathologically confined subset of ET cases with high tau pathology are identical to AD and PART. This could facilitate more precise diagnosis and therapy for ET patients with cognitive impairment.

Ancestral Sequence Reconstruction to Enable Biocatalytic Synthesis of Azaphilones.

Biocatalysis can be powerful in organic synthesis but is often limited by enzymes' substrate scope and selectivity. Developing a biocatalytic step involves identifying an initial enzyme for the target reaction followed by optimization through rational design, directed evolution, or both. These steps are time consuming, resource-intensive, and require expertise beyond typical organic chemistry. Thus, an effective strategy for streamlining the process from enzyme identification to implementation is essential to expanding biocatalysis. Here, we present a strategy combining bioinformatics-guided enzyme mining and ancestral sequence reconstruction (ASR) to resurrect enzymes for biocatalytic synthesis. Specifically, we achieve an enantioselective synthesis of azaphilone natural products using two ancestral enzymes: a flavin-dependent monooxygenase (FDMO) for stereodivergent oxidative dearomatization and a substrate-selective acyltransferase (AT) for the acylation of the enzymatically installed hydroxyl group. This cascade, stereocomplementary to established chemoenzymatic routes, expands access to enantiomeric linear tricyclic azaphilones. By leveraging the co-occurrence and coevolution of FDMO and AT in azaphilone biosynthetic pathways, we identified an AT candidate, CazE, and addressed its low solubility and stability through ASR, obtaining a more soluble, stable, promiscuous, and reactive ancestral AT (AncAT). Sequence analysis revealed AncAT as a chimeric composition of its descendants with enhanced reactivity likely due to ancestral promiscuity. Flexible receptor docking and molecular dynamics simulations showed that the most reactive AncAT promotes a reactive geometry between substrates. We anticipate that our bioinformatics-guided, ASR-based approach can be broadly applied in target-oriented synthesis, reducing the time required to develop biocatalytic steps and efficiently access superior biocatalysts.