Synthesis and Macrodomain Binding of Gln-carba-ADPr-peptide.

Mono-ADP-ribosylation is a dynamic post-translational modification (PTM) with important roles in cell signalling. This modification occurs on a wide variety of amino acids, and one of the canonical modification sites within proteins is the side chain of glutamic acid. Given the transient nature of this modification (acylal linkage) and the high sensitivity of ADP-ribosylated glutamic acid, stabilized isosteres are required for structural and biochemical studies. Here, we report the synthesis of a mimic of ADP-ribosylated peptide derived from histone H2B that contains carba-ADP-ribosylated glutamine as a potential mimic for Glu-ADPr. We synthesized a cyclopentitol-ribofuranosyl derivative of 5'-phosphoribosylated Fmoc-glutamine and used this in the solid-phase synthesis of the carba-ADPr-peptide mimicking the ADP-ribosylated N-terminal tail of histone H2B. Binding studies with isothermal calorimetry demonstrate that the macrodomains of human MacroD2 and TARG1 bind to carba-ADPr-peptide in the same way as ADPr-peptides containing the native ADP-riboside moiety connected to the side chain of glutamine in the same peptide sequence.

ICON 2020-International Scientific Tendinopathy Symposium Consensus: the development of a core outcome set for gluteal tendinopathy.

Gluteal tendinopathy (GT) is common and can be debilitating and challenging to manage. A lack of condition specific and appropriate outcome measures compromise evidence synthesis for treatment and limits clinical guideline development. Our objective was to develop a core outcome measurement set for GT (COS-GT). Participants were patients with GT and expert health professionals (HPs). A scoping review identified measures used in GT research, which were mapped to the nine International Scientific Tendinopathy Symposium Consensus core domains, and included in two surveys of HPs. The first survey identified the feasible and true measures for each domain. The second survey refined the list which a patient focus group then considered. Meeting online, HPs reached consensus (agreement ≥70%) on the most appropriate COS-GT measures. 34 HPs and seven patients were recruited. 57 measures were mapped to the nine core domains. Six measures did not proceed past survey one. Of those that progressed, none had adequate clinimetric properties for a COS-GT. Thus, participants decided on interim measures: the global rating of change, pain at night, time to pain onset with single limb stance, pain with stair walking, pain self-efficacy and hip abduction strength. HP participants additionally recommended that pain over the last week, the European Quality of Life-5 dimensions-5 levels and the Victorian Institute of Sport Assessment-Gluteal be considered in clinical trials, as they currently provide best easures of the relevant tendinopathy domains. In conclusion this interim COS-GT should guide outcome measure selection in clinical practice and future research trials in patients with GT.

Sex-Specific Implant Fixation Can Reduce Revision Rates in Total Hip Arthroplasty: Evidence From the Swiss National Joint Registry.

BACKGROUND: Patient's sex is considered a risk factor for revision following primary total hip arthroplasty (THA), but sex-specific treatment guidelines are lacking. The purpose was to assess sex-specificity of risk factors for periprosthetic femoral fractures (PFFs) and aseptic stem loosening (ASL) in a nationwide register study. METHODS: All uncemented and hybrid THAs for hip osteoarthritis registered in the Swiss National Joint Registry were considered. 86,423 THAs were analyzed. Comparable THA subsets for both sexes were obtained through propensity score matching (1:1). A sex-specific analysis of risk factors for early PFF or ASL was performed using recursive partitioning analyses. RESULTS: In women, PFFs were most significantly associated with uncemented THA fixation (P < .0001) and age (P < .01, threshold: 70.5 years). The ASLs were solely associated with patient age of <65 years (P = .023). In men, PFFs were associated exclusively with an American Society of Anesthesiologists (ASA) score >2 (P = .026). The ASLs were not correlated to any of the potential risk factors analyzed. A mathematical simulation indicated that avoiding uncemented THA fixation in women ≥70.5 years of age decreased the number of revisions within the observational period by 21% in this subset and by 4.9% in the entire patient population. CONCLUSION: Uncemented THA should be avoided in women >70.5 years due to the increased risk of early PFF, while the mode of stem fixation did not influence revision risk in men. A sex-specific regimen for THA fixation has the potential to markedly reduce early THA revision rates.

Disordered protein regions partner up the cBAF remodeler for a chromatin dance.

Intrinsically disordered protein regions form condensates and mediate interactions with factors that regulate gene activity. Patil et al.1 decode how such regions within the chromatin remodeler cBAF choreograph self-condensation and non-self interactions with transcriptional regulators, potentially impacting disease.

The histone chaperone ANP32B regulates chromatin incorporation of the atypical human histone variant macroH2A.

All vertebrate genomes encode for three large histone H2A variants that have an additional metabolite-binding globular macrodomain module, macroH2A. MacroH2A variants impact heterochromatin organization and transcription regulation and establish a barrier for cellular reprogramming. However, the mechanisms of how macroH2A is incorporated into chromatin and the identity of any chaperones required for histone deposition remain elusive. Here, we develop a split-GFP-based assay for chromatin incorporation and use it to conduct a genome-wide mutagenesis screen in haploid human cells to identify proteins that regulate macroH2A dynamics. We show that the histone chaperone ANP32B is a regulator of macroH2A deposition. ANP32B associates with macroH2A in cells and in vitro binds to histones with low nanomolar affinity. In vitro nucleosome assembly assays show that ANP32B stimulates deposition of macroH2A-H2B and not of H2A-H2B onto tetrasomes. In cells, depletion of ANP32B strongly affects global macroH2A chromatin incorporation, revealing ANP32B as a macroH2A histone chaperone.

XPC-PARP complexes engage the chromatin remodeler ALC1 to catalyze global genome DNA damage repair.

Cells employ global genome nucleotide excision repair (GGR) to eliminate a broad spectrum of DNA lesions, including those induced by UV light. The lesion-recognition factor XPC initiates repair of helix-destabilizing DNA lesions, but binds poorly to lesions such as CPDs that do not destabilize DNA. How difficult-to-repair lesions are detected in chromatin is unknown. Here, we identify the poly-(ADP-ribose) polymerases PARP1 and PARP2 as constitutive interactors of XPC. Their interaction results in the XPC-stimulated synthesis of poly-(ADP-ribose) (PAR) by PARP1 at UV lesions, which in turn enables the recruitment and activation of the PAR-regulated chromatin remodeler ALC1. PARP2, on the other hand, modulates the retention of ALC1 at DNA damage sites. Notably, ALC1 mediates chromatin expansion at UV-induced DNA lesions, leading to the timely clearing of CPD lesions. Thus, we reveal how chromatin containing difficult-to-repair DNA lesions is primed for repair, providing insight into mechanisms of chromatin plasticity during GGR.

Aseptic Revision of Total Hip Arthroplasty With a Single Modular Femoral Stem and a Modified Extended Trochanteric Osteotomy-Treatment Assessment With the Forgotten Joint Score-12.

Background: Aseptic loosening is among the most common reasons for revision total hip arthroplasty (RTHA). Modular revision stems implanted through an extended trochanteric osteotomy (ETO) promise good results, but patient-reported outcome measures (PROMs) are rarely conveyed. This study used the Forgotten Joint Score-12 (FJS-12) to assess patient-reported outcome in patients who had undergone RTHA for aseptic stem loosening using a modified ETO approach with a tapered, fluted modular stem. Material and methods: A single-center analysis of aseptic RTHA was performed (2007-2019). Clinical results (range of motion, walking ability, function), radiographic results (ETO healing, stem subsidence), and PROMs (FJS-12, Harris Hip Score, European Quality of Life 5D Score) were assessed. Minimum follow-up duration was 1 year. Complications including revisions were recorded. Results: Primary outcome parameters were assessed on 72 patients (mean age 73.3 years, mean body mass index 27.6kg/m2). Additional PROMs were collected by phone interviews from 41 patients (mean follow-up 5.7 years). In 76%, leg length was restored, and a normal gait was achieved. After 1 year, the ETO was healed in 93%; subsidence occurred in 8.3% of cases. The mean FJS-12 at the final follow-up was 85.6 ± 23.6, and the respective Harris Hip Score and European Quality of Life 5D Score averaged 87 ± 17.8 and 72.9 ± 15.9. Complication and revision rates were 33.3% and 13.9%, respectively. Conclusion: Aseptic RTHA as presented here resulted in excellent PROMs in the medium term. FJS-12 score averaged 85.6 with a mean follow-up of 5.7 years. Treatment using a modular implant and a modified ETO was associated with good clinical and radiographic outcomes. Complication and revision rates were 33.3% and 13.9%, respectively.

Tibial joint line orientation has no effect on joint awareness after mechanically aligned total knee arthroplasty.

PURPOSE: Joint line orientation (JLO) plays an important role in total knee arthroplasty (TKA), but its influence on patient-reported outcomes (PROs) is unclear. The purpose of this study was to examine JLO impact as measured by the forgotten joint score (FJS-12). The hypothesis was that restoring the joint line (JL) parallel to the floor would influence joint awareness favorably, i.e., allow the patient to forget about the joint in daily living. METHODS: All computer-navigated primary TKAs using a cemented, cruciate-retaining (CR) design implanted between January 2018 and September 2019 were reviewed in this retrospective single-center analysis. Primary endpoints were: clinical [range of motion (ROM)], and patient-reported (FJS-12) and radiographical outcomes [tibia joint line angle (TJLA), hip knee axis (HKA), mechanical medial proximal tibia angle (mMPTA) as well as mechanical lateral distal femoral angle (mLDFA)]. RESULTS: Seventy-six patients (mean age: 70.3 ± 9.7 years, mean BMI: 29.7 ± 5.2 kg/m2) were included. Postoperative ROM averaged 118.7 ± 9.6°. The mean FJS-12 improved from 16.4 ± 15.3 (preoperatively) to 89.4 ± 16.9 (1-year follow-up; p < 0.001). Clinical outcomes and PROs did not correlate with JLO (p = n.s.). Cluster analysis using six measures revealed that a medially opened TJLA was associated with significantly better postoperative FJS-12. CONCLUSION: Tibial JLO was found to have no effect on PROs. Considering the JLO in the coronal plane alone probably has questionable clinical relevance. Lower limb alignment should be assessed in all three planes and correlated with the clinical outcome. LEVEL OF CLINICAL EVIDENCE: Level IV.

ADP-ribosyltransferases, an update on function and nomenclature.

ADP-ribosylation, a modification of proteins, nucleic acids, and metabolites, confers broad functions, including roles in stress responses elicited, for example, by DNA damage and viral infection and is involved in intra- and extracellular signaling, chromatin and transcriptional regulation, protein biosynthesis, and cell death. ADP-ribosylation is catalyzed by ADP-ribosyltransferases (ARTs), which transfer ADP-ribose from NAD+ onto substrates. The modification, which occurs as mono- or poly-ADP-ribosylation, is reversible due to the action of different ADP-ribosylhydrolases. Importantly, inhibitors of ARTs are approved or are being developed for clinical use. Moreover, ADP-ribosylhydrolases are being assessed as therapeutic targets, foremost as antiviral drugs and for oncological indications. Due to the development of novel reagents and major technological advances that allow the study of ADP-ribosylation in unprecedented detail, an increasing number of cellular processes and pathways are being identified that are regulated by ADP-ribosylation. In addition, characterization of biochemical and structural aspects of the ARTs and their catalytic activities have expanded our understanding of this protein family. This increased knowledge requires that a common nomenclature be used to describe the relevant enzymes. Therefore, in this viewpoint, we propose an updated and broadly supported nomenclature for mammalian ARTs that will facilitate future discussions when addressing the biochemistry and biology of ADP-ribosylation. This is combined with a brief description of the main functions of mammalian ARTs to illustrate the increasing diversity of mono- and poly-ADP-ribose mediated cellular processes.

Mid-term outcomes following transfemoral revision of total hip arthroplasty for Vancouver B2/B3 periprosthetic fractures.

INTRODUCTION: Periprosthetic femoral fractures (PFF) are often the reason for revising total hip arthroplasty (RTHA). Converting these fractures into modified extended trochanteric osteotomy (mETO) facilitates stem revision. Modular revision stems are a common choice with good results. We present mid-term outcomes in patients undergoing RTHA for Vancouver B2/B3 PFF using a tapered, fluted modular stem with an mETO approach. MATERIALS AND METHODS: A single-center analysis of patients with RTHA for Vancouver B2/B3 PFF using a single modular implant with mETO was performed (2007 - 2019). Clinical outcome (mobility, range of motion, function), radiological outcome (fracture healing, stem subsidence) and patient reported outcome measures (FJS-12, HHS, EQ-5D) were assessed. RESULTS: Ninety-seven patients (mean age 78.1 years, BMI 25.8 kg/m2, 85.6% B2-fractures) were included; 80 patients had complete clinical and radiological follow-ups. Normal unaided gait without limping was achieved in 38/80 patients. After one year fracture / mETO healing occurred in 74/80 patients; 5.3 years after surgery, the respective FJS-12, HHS and EQ-5D (available in 34 patients) averaged 81.3 ± 30.2, 71.4 ± 18.7 and 0.8 ± 0.2. We documented 7 in-hospital deaths, 18.8% postoperative complications and 13.8% revisions with stem revisions being most commonly for subsequent PFF or subsidence. CONCLUSION: Good clinical and radiological outcomes and rather low complication and revision rates (18.8% and 13.8%) were achieved. FJS-12 showed excellent results in patients undergoing RTHA for Vancouver B2/B3 PFF using a cementless, dual modular titanium revision stem and an mETO approach. Joint awareness was thereby similar to previously reported primary THA results at 5.3 years follow-up.

Evolution of a histone variant involved in compartmental regulation of NAD metabolism.

NAD metabolism is essential for all forms of life. Compartmental regulation of NAD+ consumption, especially between the nucleus and the mitochondria, is required for energy homeostasis. However, how compartmental regulation evolved remains unclear. In the present study, we investigated the evolution of the macrodomain-containing histone variant macroH2A1.1, an integral chromatin component that limits nuclear NAD+ consumption by inhibiting poly(ADP-ribose) polymerase 1 in vertebrate cells. We found that macroH2A originated in premetazoan protists. The crystal structure of the macroH2A macrodomain from the protist Capsaspora owczarzaki allowed us to identify highly conserved principles of ligand binding and pinpoint key residue substitutions, selected for during the evolution of the vertebrate stem lineage. Metabolic characterization of the Capsaspora lifecycle suggested that the metabolic function of macroH2A was associated with nonproliferative stages. Taken together, we provide insight into the evolution of a chromatin element involved in compartmental NAD regulation, relevant for understanding its metabolism and potential therapeutic applications.

The histone chaperone FACT facilitates heterochromatin spreading by regulating histone turnover and H3K9 methylation states.

Heterochromatin formation requires three distinct steps: nucleation, self-propagation (spreading) along the chromosome, and faithful maintenance after each replication cycle. Impeding any of those steps induces heterochromatin defects and improper gene expression. The essential histone chaperone FACT (facilitates chromatin transcription) has been implicated in heterochromatin silencing, but the mechanisms by which FACT engages in this process remain opaque. Here, we pinpoint its function to the heterochromatin spreading process in fission yeast. FACT impairment reduces nucleation-distal H3K9me3 and HP1/Swi6 accumulation at subtelomeres and derepresses genes in the vicinity of heterochromatin boundaries. FACT promotes spreading by repressing heterochromatic histone turnover, which is crucial for the H3K9me2 to me3 transition that enables spreading. FACT mutant spreading defects are suppressed by removal of the H3K9 methylation antagonist Epe1. Together, our study identifies FACT as a histone chaperone that promotes heterochromatin spreading and lends support to the model that regulated histone turnover controls the propagation of repressive methylation marks.

Chemical synthesis of linear ADP-ribose oligomers up to pentamer and their binding to the oncogenic helicase ALC1.

ADP-ribosylation is a pivotal post-translational modification that mediates various important cellular processes producing negatively charged biopolymer, poly (ADP-ribose), the functions of which need further elucidation. Toward this end, the availability of well-defined ADP-ribose (ADPr) oligomers in sufficient quantities is a necessity. In this work, we demonstrate the chemical synthesis of linear ADPr oligomers of defined, increasing length using a modified solid phase synthesis method. An advanced phosphoramidite building block temporarily protected with the base sensitive Fm-group was designed and implemented in the repeating pyrophosphate formation via a P(v)-P(iii) coupling procedure on Tentagel solid support. Linear ADPr oligomers up to a pentamer were successfully synthesized and their affinity for the poly-(ADP-ribose)-binding macrodomain of the human oncogenic helicase and chromatin remodeling enzyme ALC1 was determined. Our data reveal a length-dependent binding manner of the nucleic acid, with larger ADPr oligomers exhibiting higher binding enthalpies for ALC1, illustrating how the activity of this molecular machine is gated by PAR.

Different outcomes after proximal femoral replacement in oncologic and failed revision arthroplasty patients - a retrospective cohort study.

BACKGROUND: Proximal femoral replacement (PFR) is a technically demanding procedure commonly performed to restore extensive, oncological or non-oncological bone defects in a severely debilitated patient collective. Depending on different indications, a varying outcome has been reported. The aim of the study was to assess the functional outcomes and complication rates of PFR with the modular Munich-Luebeck (MML) femoral megaprosthesis (ESKA/Orthodynamics, Luebeck, Germany), and to highlight outcome differences in patients treated for failed revision total hip arthroplasty (THA) or malignant bone disease. METHODS: A retrospective review of patients treated with PFR for failed THA or malignant tumor disease between 2000 and 2012 was performed. Patient satisfaction, functional outcome (VAS, SF-12, MSTS, WOMAC, TESS), complications and failure types (Henderson's failure classification) were assessed. A Kaplan-Meier analysis determined implant survival. RESULTS: Fifty-eight patients (age: 69.9 years, BMI: 26.7 kg/m2, mean follow-up: 66 months) were included. The mean SF-12 (physical / mental) was 37.9 / 48.4. MSTS averaged 68% at final follow-up, while mean WOMAC and TESS scored 37.8 and 59.5. TESS and WOMAC scores demonstrated significantly worse outcomes in the revision group (RG) compared to the tumor group (TG). Overall complication rate was 43.1%, and dislocation was the most common complication (27.6%). Implant survival rates were 83% (RG) and 85% (TG; p = n.s.) at 5 years, while 10-year survival was 57% (RG) and 85% (TG, p < 0.05). CONCLUSIONS: PFR is a salvage procedure for restoration of mechanical integrity and limb preservation after extensive bone loss. Complications rates are considerably high. Functional outcomes and 10-year implant survival rate were worse in the RG compared to the TG. Strict indications and disease-specific patient education are essential in preoperative planning and prognosis.

Treatment of Gluteal Tendinopathy: A Systematic Review and Stage-Adjusted Treatment Recommendation.

BACKGROUND: Gluteal tendinopathy is the most common lower limb tendinopathy. It presents with varying severity but may cause debilitating lateral hip pain. PURPOSE: To review the therapeutic options for different stages of gluteal tendinopathy, to highlight gaps within the existing evidence, and to provide guidelines for a stage-adjusted therapy for gluteal tendinopathy. STUDY DESIGN: Systematic review; Level of evidence, 4. METHODS: We screened Scopus, Embase, Web of Science, PubMed, PubMed Central, Ovid MEDLINE, CINAHL, UpToDate, and Google Scholar databases and databases for grey literature. Patient selection, diagnostic criteria, type and effect of a therapeutic intervention, details regarding aftercare, outcome assessments, complications of the treatment, follow-up, and conclusion of the authors were recorded. An assessment of study methodological quality (type of study, level of evidence) was also performed. Statistical analysis was descriptive. Data from multiple studies were combined if they were obtained from a single patient population. Weighted mean and range calculations were performed. RESULTS: A total of 27 studies (6 randomized controlled trials) with 1103 patients (1106 hips) were included. The mean age was 53.7 years (range, 17-88 years), and the mean body mass index was 28.3. The ratio of female to male patients was 7:1. Radiological confirmation of the diagnosis was most commonly obtained using magnetic resonance imaging. Reported treatment methods were physical therapy/exercise; injections (corticosteroids, platelet-rich plasma, autologous tenocytes) with or without needle tenotomy/tendon fenestration; shockwave therapy; therapeutic ultrasound; and surgical procedures such as bursectomy, iliotibial band release, and endoscopic or open tendon repair (with or without tendon augmentation). CONCLUSION: There was good evidence for using platelet-rich plasma in grades 1 and 2 tendinopathy. Shockwave therapy, exercise, and corticosteroids showed good outcomes, but the effect of corticosteroids was short term. Bursectomy with or without iliotibial band release was a valuable treatment option in grades 1 and 2 tendinopathy. Insufficient evidence was available to provide guidelines for the treatment of partial-thickness tears. There was low-level evidence to support surgical repair for grades 3 (partial-thickness tears) and 4 (full-thickness tears) tendinopathy. Fatty degeneration, atrophy, and retraction can impair surgical repair, while their effect on patient outcomes remains controversial.

Crosstalk between Drp1 phosphorylation sites during mitochondrial remodeling and their impact on metabolic adaptation.

Mitochondria constantly undergo fusion and fission events, referred as mitochondrial dynamics, which determine mitochondrial architecture and bioenergetics. Cultured cell studies demonstrate that mitochondrial dynamics are acutely regulated by phosphorylation of the mitochondrial fission orchestrator dynamin-related protein 1 (Drp1) at S579 or S600. However, the physiological impact and crosstalk of these phosphorylation sites is poorly understood. Here, we describe the functional interrelation between S579 and S600 phosphorylation sites in vivo and their role on mitochondrial remodeling. Mice carrying a homozygous Drp1 S600A knockin (Drp1 KI) mutation display larger mitochondria and enhanced lipid oxidation and respiratory capacities, granting improved glucose tolerance and thermogenic response upon high-fat feeding. Housing mice at thermoneutrality blunts these differences, suggesting a role for the brown adipose tissue in the protection of Drp1 KI mice against metabolic damage. Overall, we demonstrate crosstalk between Drp1 phosphorylation sites and provide evidence that their modulation could be used in the treatment and prevention of metabolic diseases.

Nucleolar release of rDNA repeats for repair involves SUMO-mediated untethering by the Cdc48/p97 segregase.

Ribosomal RNA genes (rDNA) are highly unstable and susceptible to rearrangement due to their repetitive nature and active transcriptional status. Sequestration of rDNA in the nucleolus suppresses uncontrolled recombination. However, broken repeats must be first released to the nucleoplasm to allow repair by homologous recombination. Nucleolar release of broken rDNA repeats is conserved from yeast to humans, but the underlying molecular mechanisms are currently unknown. Here we show that DNA damage induces phosphorylation of the CLIP-cohibin complex, releasing membrane-tethered rDNA from the nucleolus in Saccharomyces cerevisiae. Downstream of phosphorylation, SUMOylation of CLIP-cohibin is recognized by Ufd1 via its SUMO-interacting motif, which targets the complex for disassembly through the Cdc48/p97 chaperone. Consistent with a conserved mechanism, UFD1L depletion in human cells impairs rDNA release. The dynamic and regulated assembly and disassembly of the rDNA-tethering complex is therefore a key determinant of nucleolar rDNA release and genome integrity.