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.

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.