Arthroscopic Shaver-based Harvest of Minced Cartilage Results in Reduced Chondrocyte Viability and Reduced Quality of Cartilaginous Repair Tissue Compared With Open Harvest and Conventional Fragmentation.

PURPOSE: To characterize and compare the quality of regenerative cartilage tissue (ReCT) after conventional minced cartilage (CMC) and arthroscopic minced cartilage (AMC), in terms of cell viability, gene expression, and matrix synthesis and to investigate the influence of different shaver types. METHODS: Chondral tissue was harvested from the knees of 8 porcine donors. Porcine specimens were euthanized one day before harvest. AMC was created with 2 shaver blades in 2 operating modes (oscillating vs forward) and compared with a scalpel-fragmented CMC control. Before histologic analysis, 50% of the tissue was digested to prevent dedifferentiation of chondrocytes to fibroblasts. Cells were cultured and analyzed for cell viability, gene expression of cartilage-specific markers (aggrecan [ACAN], collagen type II, alpha1 [COL2A1], collagen type I, alpha1 [COL1A1], fibronectin-1 [FN1]), and matrix synthesis (Alcian-blue). RESULTS: AMC tissue contained fewer viable chondrocytes (41%-54% vs 91%; P = .001-.048) compared with CMC. After culture, CMC showed greater expressions of ACAN (27 virtual copy numbers [VCN]/housekeeping gene) and COL2A1 (30 VCN) compared with AMC (ACAN 2-9 VCN, COL2A1 2-7 VCN, P = .001-.039). AMC presented greater expressions of COL1A1 (9-21 VCN) and FN1 (12-17 VCN) than CMC (1 and 6 VCN, P = .001-.050). The signal intensity of the cartilage matrix formed by CMC (86/mm2) was greater than by AMC (7-10 mm2, P = .001-.032). CONCLUSIONS: CMC contained high numbers of viable chondrocytes, resulting in high-quality, hyaline-like ReCT. In contrast, AMC showed impaired chondrocyte quantity and viability, showing greater expressions of fibroblast markers and a decreased formation of mature cartilage matrix in porcine samples. The high chondrogenic potential of CMC to form hyaline-like ReCT was not confirmed for AMC. CLINICAL RELEVANCE: On the basis of our findings, arthroscopic harvest of minced cartilage leads to reduced chondrocyte viability and ReCT quality. Accordingly, CMC and AMC cannot be regarded as synonymous techniques, as arthroscopic techniques seem to be less efficacious.

ACL reconstruction demonstrates better stability compared to ACL repair for patients with Schenck III and IV knee dislocations.

PURPOSE: This study compared mid-term outcomes of two anterior cruciate ligament (ACL) restoration techniques within an early total surgical care of acute knee dislocation: repair with additional internal bracing (ACLIB) and reconstruction with autograft (ACLR). Initial results at 12 months demonstrated that ACLR offered superior stability compared to ACLIB. METHODS: Retrospective clinical study of patients with acute type III or IV KD. ACLIB or ACLR procedures were performed accompanied by simultaneous suture and internal bracing of the posterior cruciate ligament (PCL) and repair with lateral augmentation of the medial and lateral complex injuries utilizing Arciero's reconstruction technique. Patient-reported outcome measurements (PROMs), instrumental stability assessment via the Rolimeter-Test, and stress radiographs were analyzed. RESULTS: The study involved 20 patients (5 IIIM, 5 IIIL, and 10 IV injuries) with an average follow-up of 35.2 ± 7.4 months. Notable differences in anterior tibial translation on stress radiography favouring ACLR persisted at 24-month follow-up in side-to-side difference (SSD) (ACLIB 2.8 ± 2.5 mm vs. ACLR 0.3 ± 2.6 mm; p = 0.0487), but Rolimeter test variance diminished (SSD ACLIB 2.5 ± 0.9 mm vs. ACLR 1.8 ± 1.7 mm). Both groups showed excellent PROMs (Lysholm Score: ACLIB 84.4 ± 15.8 vs. ACLR 89.9 ± 9.0; IKDC Score: ACLIB 77.1 ± 16.2 vs. ACLR 77.7 ± 8.6). CONCLUSION: Our results indicate improved anterior stability at 12 months, which persisted at 24 months after ACL reconstruction compared with ACL repair by stress radiography. Both groups showed favourable patient-reported outcomes throughout the follow-up period. Notable rates of postoperative knee stiffness were observed in both groups. These were successfully managed with early, one-time arthroscopic arthrolysis within the first seven months of treatment, resulting in no major range of motion limitations at the 24-month follow-up. LEVEL OF EVIDENCE: Retrospective cohort study, III.

The ribosomal protein L22 binds the MDM4 pre-mRNA and promotes exon skipping to activate p53 upon nucleolar stress.

The tumor suppressor p53 and its antagonists MDM2 and MDM4 integrate stress signaling. For instance, dysbalanced assembly of ribosomes in nucleoli induces p53. Here, we show that the ribosomal protein L22 (RPL22; eL22), under conditions of ribosomal and nucleolar stress, promotes the skipping of MDM4 exon 6. Upon L22 depletion, more full-length MDM4 is maintained, leading to diminished p53 activity and enhanced cellular proliferation. L22 binds to specific RNA elements within intron 6 of MDM4 that correspond to a stem-loop consensus, leading to exon 6 skipping. Targeted deletion of these intronic elements largely abolishes L22-mediated exon skipping and re-enables cell proliferation, despite nucleolar stress. L22 also governs alternative splicing of the L22L1 (RPL22L1) and UBAP2L mRNAs. Thus, L22 serves as a signaling intermediate that integrates different layers of gene expression. Defects in ribosome synthesis lead to specific alternative splicing, ultimately triggering p53-mediated transcription and arresting cell proliferation.

Linear Factor Analytic Thurstonian Forced-Choice Models: Current Status and Issues.

Thurstonian forced-choice modeling is considered to be a powerful new tool to estimate item and person parameters while simultaneously testing the model fit. This assessment approach is associated with the aim of reducing faking and other response tendencies that plague traditional self-report trait assessments. As a result of major recent methodological developments, the estimation of normative trait scores has become possible in addition to the computation of only ipsative scores. This opened up the important possibility of comparisons between individuals with forced-choice assessment procedures. With item response theory (IRT) methods, a multidimensional forced-choice (MFC) format has also been proposed to estimate individual scores. Customarily, items to assess different traits are presented in blocks, often triplets, in applications of the MFC, which is an efficient form of item presentation but also a simplification of the original models. The present study provides a comprehensive review of the present status of Thurstonian forced-choice models and their variants. Critical features of the current models, especially the block models, are identified and discussed. It is concluded that MFC modeling with item blocks is highly problematic and yields biased results. In particular, the often-recommended presentation of blocks with items that are keyed in different directions of a trait proves to be counterproductive considering the goal to reduce response tendencies. The consequences and implications of the highlighted issues are further discussed.

Analysis of postoperative complications 5 years after osteosynthesis of patella fractures-a retrospective, multicenter cohort study.

PURPOSE: The study aims to investigate the influence of patient- and fracture-specific factors on the occurrence of complications after osteosynthesis of patella fractures and to compare knee joint function, activity, and subjective pain levels after a regular postoperative course and after complications in the medium term. METHODS: This retrospective, multicenter cohort study examined patients who received surgery for patella fracture at level 1 trauma centers between 2013 and 2018. Patient demographics and fracture-specific variables were evaluated. Final follow-up assessments included patient-reported pain scores (NRS), subjective activity and knee function scores (Tegner Activity Scale, Lysholm score, IKDC score), complications, and revisions. RESULTS: A total of 243 patients with a mean follow-up of 63.4 ± 21.3 months were included. Among them, 66.9% of patients underwent tension band wiring (TBW), 19.0% received locking plate osteosynthesis (LPO), and 14.1% underwent screw osteosynthesis (SO). A total of 38 patients (15.6%) experienced complications (TBW: 16.7%; LPO: 15.2%; SO: 11.8%). Implant-related complications of atraumatic fragment dislocation and material insufficiency/dislocation, accounted for 50% of all complications, were significantly more common after TBW than LPO (p = 0.015). No patient-specific factor was identified as a general cause for increased complications. Overall, particularly following complications such as limited range of motion or traumatic refracture, functional knee scores were significantly lower and pain levels were significantly higher at the final follow-up when a complication occurred. Implant-related complications, however, achieved functional scores comparable to a regular postoperative course without complications after revision surgery. CONCLUSION: The present study demonstrated that implant-related complications occurred significantly more often after TBW compared to LPO. The complication rates were similar in all groups.

How Natural Enzymes and Synthetic Ribozymes Generate Methylated Nucleotides in RNA.

Methylation of RNA nucleotides represents an important layer of gene expression regulation, and perturbation of the RNA methylome is associated with pathophysiology. In cells, RNA methylations are installed by RNA methyltransferases (RNMTs) that are specialized to catalyze particular types of methylation (ribose or different base positions). Furthermore, RNMTs must specifically recognize their appropriate target RNAs within the RNA-dense cellular environment. Some RNMTs are catalytically active alone and achieve target specificity via recognition of sequence motifs and/or RNA structures. Others function together with protein cofactors that can influence stability, S-adenosyl-L-methionine binding, and RNA affinity as well as aiding specific recruitment and catalytic activity. Association of RNMTs with guide RNAs represents an alternative mechanism to direct site-specific methylation by an RNMT that lacks intrinsic specificity. Recently, ribozyme-catalyzed methylation of RNA has been achieved in vitro, and here, we compare these different strategies for RNA methylation from structural and mechanistic perspectives.

Atomic Mutagenesis of N6-Methyladenosine Reveals Distinct Recognition Modes by Human m6A Reader and Eraser Proteins.

N6-methyladenosine (m6A) is an important modified nucleoside in cellular RNA associated with multiple cellular processes and is implicated in diseases. The enzymes associated with the dynamic installation and removal of m6A are heavily investigated targets for drug research, which requires detailed knowledge of the recognition modes of m6A by proteins. Here, we use atomic mutagenesis of m6A to systematically investigate the mechanisms of the two human m6A demethylase enzymes FTO and ALKBH5 and the binding modes of YTH reader proteins YTHDF2/DC1/DC2. Atomic mutagenesis refers to atom-specific changes that are introduced by chemical synthesis, such as the replacement of nitrogen by carbon atoms. Synthetic RNA oligonucleotides containing site-specifically incorporated 1-deaza-, 3-deaza-, and 7-deaza-m6A nucleosides were prepared by solid-phase synthesis and their RNA binding and demethylation by recombinant proteins were evaluated. We found distinct differences in substrate recognition and transformation and revealed structural preferences for the enzymatic activity. The deaza m6A analogues introduced in this work will be useful probes for other proteins in m6A research.

Variants in tropomyosins TPM2 and TPM3 causing muscle hypertonia.

Patients with myopathies caused by pathogenic variants in tropomyosin genes TPM2 and TPM3 usually have muscle hypotonia and weakness, their muscle biopsies often showing fibre size disproportion and nemaline bodies. Here, we describe a series of patients with hypercontractile molecular phenotypes, high muscle tone, and mostly non-specific myopathic biopsy findings without nemaline bodies. Three of the patients had trismus, whilst in one patient, the distal joints of her fingers flexed on extension of the wrists. In one biopsy from a patient with a rare TPM3 pathogenic variant, cores and minicores were observed, an unusual finding in TPM3-caused myopathy. The variants alter conserved contact sites between tropomyosin and actin.

Genomic description of acquired fluconazole- and echinocandin-resistance in patients with serial Candida glabrata isolates.

Candida glabrata is one of the most common causes of systemic candidiasis, often resistant to antifungal medications. To describe the genomic context of emerging resistance, we conducted a retrospective analysis of 82 serially collected isolates from 33 patients from population-based candidemia surveillance in the United States. We used whole-genome sequencing to determine the genetic relationships between isolates obtained from the same patient. Phylogenetic analysis demonstrated that isolates from 29 patients were clustered by patient. The median SNPs between isolates from the same patient was 30 (range: 7-96 SNPs), while unrelated strains infected four patients. Twenty-one isolates were resistant to echinocandins, and 24 were resistant to fluconazole. All echinocandin-resistant isolates carried a mutation either in the FKS1 or FKS2 HS1 region. Of the 24 fluconazole-resistant isolates, 17 (71%) had non-synonymous polymorphisms in the PDR1 gene, which were absent in susceptible isolates. In 11 patients, a genetically related resistant isolate was collected after recovering susceptible isolates, indicating in vivo acquisition of resistance. These findings allowed us to estimate the intra-host diversity of C. glabrata and propose an upper boundary of 96 SNPs for defining genetically related isolates, which can be used to assess donor-to-host transmission, nosocomial transmission, or acquired resistance. IMPORTANCE In our study, mutations associated to azole resistance and echinocandin resistance were detected in Candida glabrata isolates using a whole-genome sequence. C. glabrata is the second most common cause of candidemia in the United States, which rapidly acquires resistance to antifungals, in vitro and in vivo.

GPATCH4 regulates rRNA and snRNA 2'-O-methylation in both DHX15-dependent and DHX15-independent manners.

Regulation of RNA helicase activity, often accomplished by protein cofactors, is essential to ensure target specificity within the complex cellular environment. The largest family of RNA helicase cofactors are the G-patch proteins, but the cognate RNA helicases and cellular functions of numerous human G-patch proteins remain elusive. Here, we discover that GPATCH4 is a stimulatory cofactor of DHX15 that interacts with the DEAH box helicase in the nucleolus via residues in its G-patch domain. We reveal that GPATCH4 associates with pre-ribosomal particles, and crosslinks to the transcribed ribosomal DNA locus and precursor ribosomal RNAs as well as binding to small nucleolar- and small Cajal body-associated RNAs that guide rRNA and snRNA modifications. Loss of GPATCH4 impairs 2'-O-methylation at various rRNA and snRNA sites leading to decreased protein synthesis and cell growth. We demonstrate that the regulation of 2'-O-methylation by GPATCH4 is both dependent on, and independent of, its interaction with DHX15. Intriguingly, the ATPase activity of DHX15 is necessary for efficient methylation of DHX15-dependent sites, suggesting a function of DHX15 in regulating snoRNA-guided 2'-O-methylation of rRNA that requires activation by GPATCH4. Overall, our findings extend knowledge on RNA helicase regulation by G-patch proteins and also provide important new insights into the mechanisms regulating installation of rRNA and snRNA modifications, which are essential for ribosome function and pre-mRNA splicing.

Experience with German Research Consortia in the Field of Chemical Biology of Native Nucleic Acid Modifications.

The chemical biology of native nucleic acid modifications has seen an intense upswing, first concerning DNA modifications in the field of epigenetics and then concerning RNA modifications in a field that was correspondingly rebaptized epitranscriptomics by analogy. The German Research Foundation (DFG) has funded several consortia with a scientific focus in these fields, strengthening the traditionally well-developed nucleic acid chemistry community and inciting it to team up with colleagues from the life sciences and data science to tackle interdisciplinary challenges. This Perspective focuses on the genesis, scientific outcome, and downstream impact of the DFG priority program SPP1784 and offers insight into how it fecundated further consortia in the field. Pertinent research was funded from mid-2015 to 2022, including an extension related to the coronavirus pandemic. Despite being a detriment to research activity in general, the pandemic has resulted in tremendously boosted interest in the field of RNA and RNA modifications as a consequence of their widespread and successful use in vaccination campaigns against SARS-CoV-2. Funded principal investigators published over 250 pertinent papers with a very substantial impact on the field. The program also helped to redirect numerous laboratories toward this dynamic field. Finally, SPP1784 spawned initiatives for several funded consortia that continue to drive the fields of nucleic acid modification.

Pertrochanteric hip fracture is associated with mobility decline and poorer physical performance 4 to 6 months post-hip fracture.

BACKGROUND: To study the effect of hip fracture type on physical performance, functional ability and change in mobility four to six months after the injury. METHODS: A total of 1331 patients out of consecutive 2052 patients aged ≥ 65 years who underwent hip fracture surgery were included in the study. Patient information was collected on admission, during hospitalization, by phone interview and at the geriatric outpatient clinic 4 to 6 months after the fracture. Of the 1331 eligible patients, Grip strength, Timed Up and Go -test (TUG), Elderly Mobility Scale (EMS), mobility change compared to pre-fracture mobility level, Basic Activities of Daily Living (BADL) and Instrumental Activities of Daily Living (IADL) were used to determine physical performance and functional ability. Logistic regression was used for the analyses which was adjusted for gender, age, American Society of Anesthesiologists score, diagnosis of cognitive disorder, pre-fracture living arrangements, mobility and need of mobility aid. RESULTS: Patients with pertrochanteric hip fracture had an EMS lower than 14 (Odds Ratio (OR) 1.38, 95% confidence intervals (CI) 1.00-1.90), TUG time ≥ 20 s (OR 1.69, 95% CI 1.22-2.33) and they had declined in mobility (OR 1.58, 95% CI 1.20-2.09) compared to femoral neck fracture patients 4 to 6 months post-hip fracture in multivariable-adjusted logistic regression analyses. Grip strength and functional ability (IADL, BADL) 4 to 6 months after hip fracture did not differ between fracture types. There were no statistically significant differences in physical performance in patients with a subtrochanteric fracture compared to patients with a femoral neck fracture. CONCLUSIONS: Pertrochanteric hip fracture independently associated with poorer physical performance 4 to 6 months post hip fracture compared to other hip fracture types. Pertrochanteric hip fracture patients should be given special attention in terms of regaining their previous level of mobility.

Effect of Expansion Media on Functional Characteristics of Bone Marrow-Derived Mesenchymal Stromal Cells.

The therapeutic efficacy of mesenchymal stromal cells (MSCs) has been shown to rely on their immunomodulatory and regenerative properties. In order to obtain sufficient numbers of cells for clinical applications, MSCs have to be expanded ex vivo. Expansion media with xenogeneic-free (XF) growth-promoting supplements like human platelet lysate (PL) or serum- and xenogeneic-free (SF/XF) formulations have been established as safe and efficient, and both groups provide different beneficial qualities. In this study, MSCs were expanded in XF or SF/XF media as well as in mixtures thereof. MSCs cultured in these media were analyzed for phenotypic and functional properties. MSC expansion was optimal with SF/XF conditions when PL was present. Metabolic patterns, consumption of growth factors, and secretome of MSCs differed depending on the type and concentration of supplement. The lactate per glucose yield increased along with a higher proportion of PL. Many factors in the supernatant of cultured MSCs showed distinct patterns depending on the supplement (e.g., FGF-2, TGFß, and insulin only in PL-expanded MSC, and leptin, sCD40L PDGF-AA only in SF/XF-expanded MSC). This also resulted in changes in cell characteristics like migratory potential. These findings support current approaches where growth media may be utilized for priming MSCs for specific therapeutic applications.

Cellular functions of eukaryotic RNA helicases and their links to human diseases.

RNA helicases are highly conserved proteins that use nucleoside triphosphates to bind or remodel RNA, RNA-protein complexes or both. RNA helicases are classified into the DEAD-box, DEAH/RHA, Ski2-like, Upf1-like and RIG-I families, and are the largest class of enzymes active in eukaryotic RNA metabolism - virtually all aspects of gene expression and its regulation involve RNA helicases. Mutation and dysregulation of these enzymes have been linked to a multitude of diseases, including cancer and neurological disorders. In this Review, we discuss the regulation and functional mechanisms of RNA helicases and their roles in eukaryotic RNA metabolism, including in transcription regulation, pre-mRNA splicing, ribosome assembly, translation and RNA decay. We highlight intriguing models that link helicase structure, mechanisms of function (such as local strand unwinding, translocation, winching, RNA clamping and displacing RNA-binding proteins) and biological roles, including emerging connections between RNA helicases and cellular condensates formed through liquid-liquid phase separation. We also discuss associations of RNA helicases with human diseases and recent efforts towards the design of small-molecule inhibitors of these pivotal regulators of eukaryotic gene expression.

Comparing the stress response using heart rate variability during real and simulated crises: a pilot study.

Medical personnel often experience stress when responding to a medical emergency. A known stress-response is a measurable reduction in heart rate variability. It is currently unknown if crisis simulation can elicit the same stress response as real clinical emergencies. We aim to compare heart rate variability changes amongst medical trainees during simulated and real medical emergencies. We performed a single center prospective observational study, enrolling 19 resident physicians. Heart rate variability was measured in real time, using a 2-lead heart rate monitor (Bodyguard 2, Firstbeat Technologies Ltd) worn during 24 h critical care call shifts. Data was collected at baseline, during crisis simulation and when responding to medical emergencies. 57 observations were made to compare participant's heart rate variability. Each heart rate variability metric changed as expected in response to stress. Statistically significant differences were observed between baseline and simulated medical emergencies in Standard Deviation of the N-N interval (SDNN), Root mean square standard deviation of the N-N interval (RMSSD), Percentage of successive R-R intervals that differ by more than 50 ms (PNN50), Low Frequency (LF) and Low Frequency: High Frequency ratios (LF:HF). No statistically significant differences between simulated and real medical emergencies were identified in any heart rate variability metrics. We have shown using objective results, that simulation can elicit the same psychophysiological response as actual medical emergencies. Therefore, simulation may represent a reasonable way to practice not only essential skills in a safe environment but has the additional benefit of creating a realistic, physiological response in medical trainees.

N 2-methylguanosine modifications on human tRNAs and snRNA U6 are important for cell proliferation, protein translation and pre-mRNA splicing.

Modified nucleotides in non-coding RNAs, such as tRNAs and snRNAs, represent an important layer of gene expression regulation through their ability to fine-tune mRNA maturation and translation. Dysregulation of such modifications and the enzymes installing them have been linked to various human pathologies including neurodevelopmental disorders and cancers. Several methyltransferases (MTases) are regulated allosterically by human TRMT112 (Trm112 in Saccharomyces cerevisiae), but the interactome of this regulator and targets of its interacting MTases remain incompletely characterized. Here, we have investigated the interaction network of human TRMT112 in intact cells and identify three poorly characterized putative MTases (TRMT11, THUMPD3 and THUMPD2) as direct partners. We demonstrate that these three proteins are active N2-methylguanosine (m2G) MTases and that TRMT11 and THUMPD3 methylate positions 10 and 6 of tRNAs, respectively. For THUMPD2, we discovered that it directly associates with the U6 snRNA, a core component of the catalytic spliceosome, and is required for the formation of m2G, the last 'orphan' modification in U6 snRNA. Furthermore, our data reveal the combined importance of TRMT11 and THUMPD3 for optimal protein synthesis and cell proliferation as well as a role for THUMPD2 in fine-tuning pre-mRNA splicing.

The Rab5 effector FERRY links early endosomes with mRNA localization.

Localized translation is vital to polarized cells and requires precise and robust distribution of different mRNAs and ribosomes across the cell. However, the underlying molecular mechanisms are poorly understood and important players are lacking. Here, we discovered a Rab5 effector, the five-subunit endosomal Rab5 and RNA/ribosome intermediary (FERRY) complex, that recruits mRNAs and ribosomes to early endosomes through direct mRNA-interaction. FERRY displays preferential binding to certain groups of transcripts, including mRNAs encoding mitochondrial proteins. Deletion of FERRY subunits reduces the endosomal localization of transcripts in cells and has a significant impact on mRNA levels. Clinical studies show that genetic disruption of FERRY causes severe brain damage. We found that, in neurons, FERRY co-localizes with mRNA on early endosomes, and mRNA loaded FERRY-positive endosomes are in close proximity of mitochondria. FERRY thus transforms endosomes into mRNA carriers and plays a key role in regulating mRNA distribution and transport.

Molecular functions of RNA helicases during ribosomal subunit assembly.

During their biogenesis, the ribosomal subunits undergo numerous structural and compositional changes to achieve their final architecture. RNA helicases are a key driving force of such remodelling events but deciphering their particular functions has long been challenging due to lack of knowledge of their molecular functions and RNA substrates. Advances in the biochemical characterisation of RNA helicase activities together with new insights into RNA helicase binding sites on pre-ribosomes and structural snapshots of pre-ribosomal complexes containing RNA helicases now open the door to a deeper understanding of precisely how different RNA helicases contribute to ribosomal subunit maturation.