CAG-Repeat RNA Hairpin Folding and Recruitment to Nuclear Speckles with a Pivotal Role of ATP as a Cosolute.

A hallmark of Huntington's disease (HD) is a prolonged polyglutamine sequence in the huntingtin protein and, correspondingly, an expanded cytosine, adenine, and guanine (CAG) triplet repeat region in the mRNA. A majority of studies investigating disease pathology were concerned with toxic huntingtin protein, but the mRNA moved into focus due to its recruitment to RNA foci and emerging novel therapeutic approaches targeting the mRNA. A hallmark of CAG-RNA is that it forms a stable hairpin in vitro which seems to be crucial for specific protein interactions. Using in-cell folding experiments, we show that the CAG-RNA is largely destabilized in cells compared to dilute buffer solutions but remains folded in the cytoplasm and nucleus. Surprisingly, we found the same folding stability in the nucleoplasm and in nuclear speckles under physiological conditions suggesting that CAG-RNA does not undergo a conformational transition upon recruitment to the nuclear speckles. We found that the metabolite adenosine triphosphate (ATP) plays a crucial role in promoting unfolding, enabling its recruitment to nuclear speckles and preserving its mobility. Using in vitro experiments and molecular dynamics simulations, we found that the ATP effects can be attributed to a direct interaction of ATP with the nucleobases of the CAG-RNA rather than ATP acting as "a fuel" for helicase activity. ATP-driven changes in CAG-RNA homeostasis could be disease-relevant since mitochondrial function is affected in HD disease progression leading to a decline in cellular ATP levels.

Intracellular spatially-targeted chemical chaperones increase native state stability of mutant SOD1 barrel.

Amyotrophic lateral sclerosis (ALS) is a progressive neurological disorder with currently no cure. Central to the cellular dysfunction associated with this fatal proteinopathy is the accumulation of unfolded/misfolded superoxide dismutase 1 (SOD1) in various subcellular locations. The molecular mechanism driving the formation of SOD1 aggregates is not fully understood but numerous studies suggest that aberrant aggregation escalates with folding instability of mutant apoSOD1. Recent advances on combining organelle-targeting therapies with the anti-aggregation capacity of chemical chaperones have successfully reduce the subcellular load of misfolded/aggregated SOD1 as well as their downstream anomalous cellular processes at low concentrations (micromolar range). Nevertheless, if such local aggregate reduction directly correlates with increased folding stability remains to be explored. To fill this gap, we synthesized and tested here the effect of 9 ER-, mitochondria- and lysosome-targeted chemical chaperones on the folding stability of truncated monomeric SOD1 (SOD1bar) mutants directed to those organelles. We found that compound ER-15 specifically increased the native state stability of ER-SOD1bar-A4V, while scaffold compound FDA-approved 4-phenylbutyric acid (PBA) decreased it. Furthermore, our results suggested that ER15 mechanism of action is distinct from that of PBA, opening new therapeutic perspectives of this novel chemical chaperone on ALS treatment.

Multicentric development and validation of a multi-scale and multi-task deep learning model for comprehensive lower extremity alignment analysis.

Osteoarthritis of the knee, a widespread cause of knee disability, is commonly treated in orthopedics due to its rising prevalence. Lower extremity misalignment, pivotal in knee injury etiology and management, necessitates comprehensive mechanical alignment evaluation via frequently-requested weight-bearing long leg radiographs (LLR). Despite LLR's routine use, current analysis techniques are error-prone and time-consuming. To address this, we conducted a multicentric study to develop and validate a deep learning (DL) model for fully automated leg alignment assessment on anterior-posterior LLR, targeting enhanced reliability and efficiency. The DL model, developed using 594 patients' LLR and a 60%/10%/30% data split for training, validation, and testing, executed alignment analyses via a multi-step process, employing a detection network and nine specialized networks. It was designed to assess all vital anatomical and mechanical parameters for standard clinical leg deformity analysis and preoperative planning. Accuracy, reliability, and assessment duration were compared with three specialized orthopedic surgeons across two distinct institutional datasets (136 and 143 radiographs). The algorithm exhibited equivalent performance to the surgeons in terms of alignment accuracy (DL: 0.21 ± 0.18°to 1.06 ± 1.3°vs. OS: 0.21 ± 0.16°to 1.72 ± 1.96°), interrater reliability (ICC DL: 0.90 ± 0.05 to 1.0 ± 0.0 vs. ICC OS: 0.90 ± 0.03 to 1.0 ± 0.0), and clinically acceptable accuracy (DL: 53.9%-100% vs OS 30.8%-100%). Further, automated analysis significantly reduced analysis time compared to manual annotation (DL: 22 ± 0.6 s vs. OS; 101.7 ± 7 s, p ≤ 0.01). By demonstrating that our algorithm not only matches the precision of expert surgeons but also significantly outpaces them in both speed and consistency of measurements, our research underscores a pivotal advancement in harnessing AI to enhance clinical efficiency and decision-making in orthopaedics.

Clinical Effect of Isolated Lateral Closing Wedge Distal Femoral Osteotomy Compared to Medial Opening Wedge High Tibial Osteotomy for the Correction of Varus Malalignment: A Propensity Score-Matched Analysis.

BACKGROUND: Recent evidence questions the role of medial opening wedge high tibial osteotomy (mowHTO) in the correction of femoral-based varus malalignment because of the potential creation of an oblique knee joint line. However, the clinical effectiveness of alternatively performing an isolated lateral closing wedge distal femoral osteotomy (lcwDFO), in which the mechanical unloading effect in knee flexion may be limited, is yet to be confirmed. PURPOSE/HYPOTHESIS: The purpose of this article was to compare clinical outcomes between patients undergoing varus correction via isolated lcwDFO or mowHTO, performed according to the location of the deformity, in a cohort matched for confounding variables. It was hypothesized that results from undergoing isolated lcwDFO for symptomatic varus malalignment would not significantly differ from the results after mowHTO. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: Consecutive patients who underwent isolated mowHTO or lcwDFO according to a tibial- or femoral-based symptomatic varus deformity between January 2010 and October 2019 were enrolled. Confounding factors, including age at surgery, sex, body mass index, preoperative femorotibial axis, and postoperative follow-up, were matched using propensity score matching. The International Knee Documentation Committee (IKDC) Subjective Knee Form, Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), Lysholm score, Tegner Activity Scale, and visual analog scale (VAS) for pain were collected preoperatively and at a minimum of 24 months postoperatively. RESULTS: Of 535 knees assessed for eligibility, 50 knees (n = 50 patients, n = 25 per group) were selected by propensity score matching. Compared with preoperatively, both the mowHTO group (IKDC, 55.1 ± 16.5 vs 71.3 ± 14.7, P = .002; WOMAC, 22.0 ± 18.0 vs 9.6 ± 10.8, P < .001; Lysholm, 55.2 ± 23.1 vs 80.7 ± 16, P < .001; VAS, 4.1 ± 2.4 vs 1.6 ± 1.8, P < .001) and the lcwDFO group (IKDC, 49.4 ± 14.6 vs 66 ± 20.1, P = .003; WOMAC, 25.2 ± 17.0 vs 12.9 ± 17.6, P = .003; Lysholm, 46.5 ± 15.6 vs 65.4 ± 28.7, P = .011; VAS, 4.5 ± 2.2 vs 2.6 ± 2.5, P = .001) had significantly improved at follow-up (80 ± 20 vs 81 ± 43 months). There were no significant differences between the groups at baseline, at final follow-up, or in the amount of clinical improvement in any of the outcome parameters (P > .05; respectively). CONCLUSION: Performing both mowHTO or lcwDFO yields significant improvement in clinical outcomes if performed at the location of the deformity of varus malalignment. These findings confirm the clinical effectiveness of performing an isolated lcwDFO in femoral-based varus malalignment, which is comparable with that of mowHTO in the correction of varus malalignment.

Strategies for Isolating and Propagating Circulating Tumor Cells in Men with Metastatic Prostate Cancer.

Selecting a well-suited method for isolating/characterizing circulating tumor cells (CTCs) is challenging. Evaluating sensitive and specific markers for prostate cancer (PCa)-specific CTC identification and analysis is crucial. We used the CellCollector EpCAM-functionalized system (CC-EpCAM) and evaluated and developed a PCa-functionalized version (CC-PCa); we then compared CTC isolation techniques that exploit the physical and biological properties of CTCs. We established two cohorts of metastatic PCa patients (mPCa; 15 in cohort 1 and 10 in cohort 2). CTC cultivation experiments were conducted with two capturing methods (Ficoll and ScreenCell). The most sensitive detection rates and highest CTC counts were reached with the CC-PCa and ScreenCell system. Patients with ≥5 CTCs isolated with CC-EpCAM had an overall survival (OS) of 0.93 years, and patients with ≥5 CTCs isolated with CC-PCa had an OS of 1.5 years in cohort 1. Nevertheless, we observed the highest sensitivity and specificity for 24-month survival by the Ficoll with CD45 depletion and ScreenCell system with May-Grunwald Giemsa (MGG) staining. The EpCAM molecule is an essential factor related to OS for CTC isolation based on biological properties in mPCa patients. The best-suited CTC capture system is not limited to one characteristic of cells but adapted to downstream analysis.