A Multilaboratory Comparison of Calibration Accuracy and the Performance of External References in Analytical Ultracentrifugation

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies

A multilaboratory comparison of calibration accuracy and the performance of external references in analytical ultracentrifugation.

Analytical ultracentrifugation (AUC) is a first principles based method to determine absolute sedimentation coefficients and buoyant molar masses of macromolecules and their complexes, reporting on their size and shape in free solution. The purpose of this multi-laboratory study was to establish the precision and accuracy of basic data dimensions in AUC and validate previously proposed calibration techniques. Three kits of AUC cell assemblies containing radial and temperature calibration tools and a bovine serum albumin (BSA) reference sample were shared among 67 laboratories, generating 129 comprehensive data sets. These allowed for an assessment of many parameters of instrument performance, including accuracy of the reported scan time after the start of centrifugation, the accuracy of the temperature calibration, and the accuracy of the radial magnification. The range of sedimentation coefficients obtained for BSA monomer in different instruments and using different optical systems was from 3.655 S to 4.949 S, with a mean and standard deviation of (4.304 ± 0.188) S (4.4%). After the combined application of correction factors derived from the external calibration references for elapsed time, scan velocity, temperature, and radial magnification, the range of s-values was reduced 7-fold with a mean of 4.325 S and a 6-fold reduced standard deviation of ± 0.030 S (0.7%). In addition, the large data set provided an opportunity to determine the instrument-to-instrument variation of the absolute radial positions reported in the scan files, the precision of photometric or refractometric signal magnitudes, and the precision of the calculated apparent molar mass of BSA monomer and the fraction of BSA dimers. These results highlight the necessity and effectiveness of independent calibration of basic AUC data dimensions for reliable quantitative studies

Insertion of Pro-Hyp-Gly provides 2 kcal mol⁻¹ stability but attenuates the specific assembly of ABC heterotrimeric collagen triple helices

Collagen triple helices are stabilized by 2 kcal mol−1 upon inserting proline-hydroxyproline-glycine but attenuates the specific assembly of the ABC heterotrimer.</p

Intrinsically disordered regions and RNA binding domains contribute to protein enrichment in biomolecular condensates in Xenopus oocytes

Abstract Proteins containing both intrinsically disordered regions (IDRs) and RNA binding domains (RBDs) can phase separate in vitro, forming bodies similar to cellular biomolecular condensates. However, how IDR and RBD domains contribute to in vivo recruitment of proteins to biomolecular condensates remains poorly understood. Here, we analyzed the roles of IDRs and RBDs in L-bodies, biomolecular condensates present in Xenopus oocytes. We show that a cytoplasmic isoform of hnRNPAB, which contains two RBDs and an IDR, is highly enriched in L-bodies. While both of these domains contribute to hnRNPAB self-association and phase separation in vitro and mediate enrichment into L-bodies in oocytes, neither the RBDs nor the IDR replicate the localization of full-length hnRNPAB. Our results suggest a model where the combined effects of the IDR and RBDs regulate hnRNPAB partitioning into L-bodies. This model likely has widespread applications as proteins containing RBD and IDR domains are common biomolecular condensate residents

Retrograde Axonal Transport: a Major Transmission Route of Enterovirus 71 in Mice▿

Inoculation of enterovirus 71 (EV71) by the oral (p.o.), intramuscular (i.m.), or intracranial route resulted in brain infection, flaccid paralysis, pulmonary dysfunction, and death of 7-day-old mice. The lag time of disease progression indicated that neuroinvasion from the inoculation sites was a prerequisite for the development of the clinical signs. Although EV71 p.o. inoculation led to a persistent viremia and a transient increase in blood-brain barrier permeability at the early stage of the infection, only low levels of virus, which led to neither severe infection nor clinical illness, could be detected in the brain, suggesting that hematogenous transport might not represent a major transmission route. In the spinal cord, following both p.o. and hind limb i.m. inoculation, the virus first appeared and increased rapidly in the lower segments, especially at the anterior horn areas, and then spread to the upper segments and brain in the presence of viremia. A reverse pattern, with the virus being first detected in the upper segment, was observed when the virus was i.m. inoculated in the forelimb. Colchicine, a fast axonal transport inhibitor, but not sciatic nerve transection reduced EV71 neuroinvasion in a dose-dependent manner, indicating a neuronal transmission of the virus

Long-Term Outcomes of Modified Expansive Open-Door Laminoplasty Combined with Short-Level Anterior Cervical Fusion in Multilevel Cervical Spondylotic Myelopathy

Background and Objectives: Multilevel cervical spondylotic myelopathy (MCSM) presents complex challenges for surgical management, particularly in patients with kyphosis or significant anterior pathology. This study aimed to assess the long-term efficacy of modified expansive open-door laminoplasty (MEOLP) combined with short-level anterior cervical fusion (ACF) in providing decompression, preserving alignment, and maintaining range of motion (ROM) over a nine-year follow-up. Materials and Methods: A retrospective analysis was conducted on 124 MCSM patients treated with MEOLP combined with ACF between 2011 and 2015. MEOLP, a muscle-sparing posterior approach, was combined with ACF to correct sagittal misalignment and address anterior compression. Key outcome measures included the Pavlov ratio, C2–C7 angle, Japanese Orthopedic Association (JOA) score, and Visual Analog Scale (VAS) for neck pain. Patients were monitored for adjacent segment degeneration (ASD) and other postoperative changes over the long-term follow-up. Results: At nine years post-surgery, patients demonstrated significant improvements in decompression and cervical alignment. The mean C2–C7 angle increased, reflecting enhanced lordotic curvature, while the Pavlov ratio showed maintained canal expansion. JOA scores improved significantly, indicating reduced myelopathy symptoms, and VAS scores for neck pain decreased, reflecting symptom relief. Despite these positive outcomes, ASD was noted, especially in patients with reduced preoperative disk height, highlighting the need for strategies to mitigate degeneration at adjacent segments. Conclusions: MEOLP combined with short-level ACF is a viable and durable option for managing complex MCSM cases, offering effective decompression, alignment correction, and ROM preservation. The limitations of this study, including its retrospective, single-center design and the lack of quality-of-life assessments, underscore the need for future multi-center studies with broader outcome measures. These findings support MEOLP with ACF as an alternative approach in cases where traditional laminoplasty may be insufficient