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The objective of our trial was to determine the efficacy of dietary soybean meal (SBM) in reducing the effect of a porcine reproductive and respiratory syndrome virus (PRRSv) challenge in nursery pigs as measured by immune response and growth performance. The 2 dietary treatments differed by method of supplying amino acids: either by SBM or synthetic amino acids (SAA) with SBM. Seventy-two mixed sex, weanling pigs (21 d of age, 10.83 ± 0.82 kg) were allotted by weight and sex to 1 of 18 pens in a completely random design. There were 4 pigs/pen and 9 observations/treatment. All pigs were fed the same industry-standard diet for 14 d, and then were fed 1 of 2 experimental diets for 10 d. All pigs were then inoculated both intramuscularly and intranasally with 1 mL each of live PRRS virus MN-184 (1× 106 fluorescent focus units (FFU)/ mL dose) at 38 d of age (0 d post-inoculation, DPI). Blood was collected on 0, 3, 7, 14, and 28 DPI for determination of serum PRRSv load and cytokine concentrations. Pig BW and pen feed intake were recorded on blood collection days for the first 28 d and then bi-weekly until the termination of the trial at 125 kg BW. Pigs in the AA group tended to have lower TNF-α (Tumor Necrosis Factor-α) and IL-8 (InterLuekin-8) concentrations (P = 0.100 and P = 0.100) respectively on 0 DPI. At 3 DPI, pigs fed SBM vs. SAA had higher ADG (0.613 vs. 0.299 kgs) (P = 0.005) and G:F (0.603 vs. 0.336) (P = 0.0007). On 3 DPI, pigs fed the AA treatment tended to have lower IL-8 concentrations (117 vs. 145 pg/mL) (P = 0.08). Serum concentrations of INF-γ (Interferon Gamma) tended to be lower for the AA group at 7 DPI. At 14 DPI, serum concentrations of IL-4 (Interleukin-4) (P = 0.025) were higher in pigs fed the SBM treatment. Pigs fed the AA diets had higher INF-γ concentrations on 14 DPI (P = 0.034) and it tended to be higher (P = 0.080) at 28 DPI. Results observed for growth performance and blood parameters were not consistent between treatments throughout the trial. While initial growth responses were improved by SBM diets, an overall increase in immune response was observed from pigs fed the AA based diets. Therefore, additional work needs to be done in clarifying the role of amino acid source in piglet growth and immune status. [ABSTRACT FROM AUTHOR]

Obesity exacerbates tissue degeneration and compromises the integrity and reparative potential of mesenchymal stem/stromal cells (MSCs), but the underlying mechanisms have not been sufficiently elucidated. Mitochondria modulate the viability, plasticity, proliferative capacity, and differentiation potential of MSCs. We hypothesized that alterations in the 5-hydroxymethylcytosine (5hmC) profile of mitochondria-related genes may mediate obesity-driven dysfunction of human adipose-derived MSCs. MSCs were harvested from abdominal subcutaneous fat of obese and age/sex-matched non-obese subjects (n = 5 each). The 5hmC profile and expression of nuclear-encoded mitochondrial genes were examined by hydroxymethylated DNA immunoprecipitation sequencing (h MeDIP-seq) and mRNA-seq, respectively. MSC mitochondrial structure (electron microscopy) and function, metabolomics, proliferation, and neurogenic differentiation were evaluated in vitro, before and after epigenetic modulation. hMeDIP-seq identified 99 peaks of hyper-hydroxymethylation and 150 peaks of hypo-hydroxymethylation in nuclear-encoded mitochondrial genes from Obese- versus Non-obese-MSCs. Integrated hMeDIP-seq/mRNA-seq analysis identified a select group of overlapping (altered levels of both 5hmC and mRNA) nuclear-encoded mitochondrial genes involved in ATP production, redox activity, cell proliferation, migration, fatty acid metabolism, and neuronal development. Furthermore, Obese-MSCs exhibited decreased mitochondrial matrix density, membrane potential, and levels of fatty acid metabolites, increased superoxide production, and impaired neuronal differentiation, which improved with epigenetic modulation. Obesity elicits epigenetic changes in mitochondria-related genes in human adipose-derived MSCs, accompanied by structural and functional changes in their mitochondria and impaired fatty acid metabolism and neurogenic differentiation capacity. These observations may assist in developing novel therapies to preserve the potential of MSCs for tissue repair and regeneration in obese individuals., (© 2024. The Author(s).)

Aims: Embolic stroke of undetermined source (ESUS) accounts for up to 20% of ischemic strokes annually. Undetected atrial fibrillation (AF) is one important potential underlying cause. For AF, oral anticoagulation has evolved as the most preferable means of secondary stroke prevention. To detect unrecognized paroxysmal AF, long-term ECG monitoring is required, and implantable cardiac monitors (ICM) appear most suitable. Yet, ICMs are particularly costly, implantation is invasive, and remote monitoring places a personnel burden on health care providers. Here, we use data from a large cohort of ESUS patients to systematically analyze the effort of ICM remote monitoring for AF diagnosis and the strain on health care providers., Methods and Results: From a prospective, single-center, observational ESUS registry, we analyzed all ICM-equipped patients post-ESUS (n = 172) between January 1st, 2018, and December 31st, 2019. Through January 2nd, 2023, 48 patients (27.9%) were diagnosed with AF by ICM remote monitoring. During follow-up, a total of 29,180 remote monitoring episodes were transmitted, of which 17,742 were alarms for AF. A systematic estimation of workload revealed that on average, 20.3 trained physician workhours are required to diagnose one patient with AF., Conclusion: ICM remote monitoring is useful to diagnose AF in cohort of post-ESUS patients. However, the number of ICM alarms is high, even in a cohort at known high risk of AF and in whom AF detection is therapeutically consequential. Improved automated event classification, clear recommendations for ICM interrogation after AF diagnosis, and a careful patient selection for ICM monitoring are warranted., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 von Falkenhausen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Arthrofibrosis, which causes joint motion restrictions, is a common complication following total knee arthroplasty (TKA). Key features associated with arthrofibrosis include myofibroblast activation, knee stiffness, and excessive scar tissue formation. We previously demonstrated that adiponectin levels are suppressed within the knee tissues of patients affected by arthrofibrosis and showed that AdipoRon, an adiponectin receptor agonist, exhibited anti-fibrotic properties in human mesenchymal stem cells. In this study, the therapeutic potential of AdipoRon was evaluated on TGFβ1-mediated myofibroblast differentiation of primary human knee fibroblasts and in a mouse model of knee stiffness. Picrosirius red staining revealed that AdipoRon reduced TGFβ1-induced collagen deposition in primary knee fibroblasts derived from patients undergoing primary TKA and revision TKA for arthrofibrosis. AdipoRon also reduced mRNA and protein levels of ACTA2, a key myofibroblast marker. RNA-seq analysis corroborated the anti-myofibrogenic effects of AdipoRon. In our knee stiffness mouse model, 6 weeks of knee immobilization, to induce a knee contracture, in conjunction with daily vehicle (DMSO) or AdipoRon (1, 5, and 25 mg/kg) via intraperitoneal injections were well tolerated based on animal behavior and weight measurements. Biomechanical testing demonstrated that passive extension angles (PEAs) of experimental knees were similar between vehicle and AdipoRon treatment groups in mice evaluated immediately following immobilization. Interestingly, relative to vehicle-treated mice, 5 mg/kg AdipoRon therapy improved the PEA of the experimental knees in mice that underwent 4 weeks of knee remobilization following the immobilization and therapy. Together, these studies revealed that AdipoRon may be an effective therapeutic modality for arthrofibrosis., (© 2023 Wiley Periodicals LLC.)

Aims: In-hospital complications of catheter ablation for atrial fibrillation (AF), atrial flutter (AFL), and ventricular tachycardia (VT) may be overestimated by analyses of administrative data., Methods and Results: We determined the incidences of in-hospital mortality, major bleeding, and stroke around AF, AFL, and VT ablations in four German tertiary centres between 2005 and 2020. All cases were coded by the G-DRG- and OPS-systems. Uniform code search terms were applied defining both the types of ablations for AF, AFL, and VT and the occurrence of major adverse events including femoral vascular complications, iatrogenic tamponade, stroke, and in-hospital death. Importantly, all complications were individually reviewed based on patient-level source records. Overall, 43 031 ablations were analysed (30 361 AF; 9364 AFL; 3306 VT). The number of ablations/year more than doubled from 2005 (n = 1569) to 2020 (n = 3317) with 3 times and 2.5 times more AF and VT ablations in 2020 (n = 2404 and n = 301, respectively) as compared to 2005 (n = 817 and n = 120, respectively), but a rather stable number of AFL ablations (n = 554 vs. n = 612). Major peri-procedural complications occurred in 594 (1.4%) patients. Complication rates were 1.1% (n = 325) for AF, 1.0% (n = 95) for AFL, and 5.3% (n = 175) for VT. With an increase in complex AF/VT procedures, the overall complication rate significantly increased (0.76% in 2005 vs. 1.81% in 2020; P = 0.004); but remained low over time. Following patient-adjudication, all in-hospital cardiac tamponades (0.7%) and strokes (0.2%) were related to ablation. Major femoral vascular complications requiring surgical intervention occurred in 0.4% of all patients. The in-hospital mortality rate adjudicated to be ablation-related was lower than the coded mortality rate: AF: 0.03% vs. 0.04%; AFL: 0.04% vs. 0.14%; VT: 0.42% vs. 1.48%., Conclusion: Major adverse events are low and comparable after catheter ablation for AFL and AF (∼1.0%), whereas they are five times higher for VT ablations. In the presence of an increase in complex ablation procedures, a moderate but significant increase in overall complications from 2005-20 was observed. Individual case analysis demonstrated a lower than coded ablation-related in-hospital mortality. This highlights the importance of individual case adjudication when analysing administrative data., Competing Interests: Conflict of interest: L.E. discloses consultant fees, speaking honoraria, and travel expenses from Abbott, Bayer Healthcare, Biosense Webster, Biotronik, Boehringer, Boston Scientific, Bristol-Myers Squibb, Daiichi Sankyo, Medtronic, Pfizer, and Sanofi Aventis. Research has been supported by German Research Foundation (DFG) and German Heart Foundation outside the submitted work. The other authors declared no conflicts of interest regarding this study., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Osteoblast differentiation is epigenetically suppressed by the H3K27 methyltransferase EZH2, and induced by the morphogen BMP2 and transcription factor RUNX2. These factors also regulate distinct G protein coupled receptors (GPRCs; e.g., PTH1R, GPR30/GPER1). Because GPRCs transduce many physiological stimuli, we examined whether BMP2 or EZH2 inhibition (i.e., GSK126) regulates other GPRC genes in osteoblasts. RNA-seq screening of >400 mouse GPRC-related genes showed that many GPRCs are downregulated during osteogenic differentiation. The orphan receptor GPRC5C, along with a small subset of other GPRCs, is induced by BMP2 or GSK126 during Vitamin C dependent osteoblast differentiation, but not by all-trans retinoic acid. ChIP-seq analysis revealed that GSK126 reduces H3K27me3 levels at the GPRC5C gene locus in differentiating MC3T3-E1 osteoblasts, consistent with enhanced GPRC5C mRNA expression. Loss of function analyses revealed that shRNA-mediated depletion of GPRC5C decreases expression of bone markers (e.g., BGLAP and IBSP) and mineral deposition in response to BMP2 or GSK126. GPRC5C mRNA was found to be reduced in the osteopenic bones of KLF10 null mice which have compromised BMP2 signaling. GPRC5C mRNA is induced by the bone-anabolic activity of 17β-estradiol in trabecular but not cortical bone following ovariectomy. Collectively, these findings suggest that GPRC5C protein is a key node in a pro-osteogenic axis that is normally suppressed by EZH2-mediated H3K27me3 marks and induced during osteoblast differentiation by GSK126, BMP2, and/or 17β-estradiol. Because GPRC5C protein is an understudied orphan receptor required for osteoblast differentiation, identification of ligands that induce GPRC5C signaling may support therapeutic strategies to mitigate bone-related disorders., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Vitamin C (VitC) is essential for bone health, and low VitC serum levels increase the risk for skeletal fractures. If and how VitC affects bone mineralization is unclear. Using micro-computed tomography (μCT), histologic staining, as well as quantitative backscattered electron imaging (qBEI), we assessed the effects of VitC on femoral structure and microarchitecture, bone formation, and bone mineralization density distribution (BMDD) in the VitC incompetent Gulo -/- mouse model and wild-type mice. In particular, VitC-supplemented, 20-week-old mice were compared with age-matched counterparts where dietary VitC intake was excluded from week 15. VitC depletion in Gulo -/- mice severely reduced cortical thickness of the diaphyseal shaft and bone volume around the growth plate (eg, bone volume of the primary spongiosa -43%, p < 0.001). Loss of VitC also diminished the amount of newly formed bone tissue as visualized by histology and calcein labeling of the active mineralization front. BMDD analysis revealed a shift to higher calcium concentrations upon VitC supplementation, including higher average (~10% increase in female VitC deficient mice, p < 0.001) and peak calcium concentrations in the epiphyseal and metaphyseal spongiosa. These findings suggest higher bone tissue age. Importantly, loss of VitC had significantly more pronounced effects in female mice, indicating a higher sensitivity of their skeleton to VitC deficiency. Our results reveal that VitC plays a key role in bone formation rate, which directly affects mineralization. We propose that low VitC levels may contribute to the higher prevalence of bone-degenerative diseases in females and suggest leveraging this vitamin against these conditions. © 2023 American Society for Bone and Mineral Research (ASBMR)., (© 2023 American Society for Bone and Mineral Research (ASBMR).)

New clathrochelate complexes of manganese, iron and cobalt containing peripheral organometallic manganese moieties cymantrene or tromancenium were synthesized via self-assembly from di/tri-topic dioximes, metal templates and cymantrene/tromancenium boronic acid pinacol esters. These air-stable, highly colored, oligometallic complexes are composed of various combinations of Mn I Fe II Mn I , Mn I Co II Mn I , Mn I Mn II Mn II Mn I and Mn I Co II Co II Mn I metal assemblies with corresponding complicated magnetic and electrochemical properties. Full spectroscopic and structural characterization by 1 H/ 11 B/ 13 C NMR, HRMS, IR, UV-vis, single crystal XRD and CV (cyclic voltammetry) is provided. Tetrametallic complexes containing tromanceniumyl substituents with two Co II or Mn II central metals exhibit promising anticancer properties against different tumor cell lines., Competing Interests: The authors declare no conflict of interest., (© 2023 The Authors. European Journal of Inorganic Chemistry published by Wiley-VCH GmbH.)

Bivalent epigenomic regulatory domains containing both activating histone 3 lysine 4 (H3K4me3) and repressive lysine 27 (H3K27me3) trimethylation are associated with key developmental genes. These bivalent domains repress transcription in the absence of differentiation signals but maintain regulatory genes in a poised state to allow for timely activation. Previous studies demonstrated that enhancer of zeste homolog 2 (Ezh2), a histone 3 lysine 27 (H3K27) methyltransferase, suppresses osteogenic differentiation and that inhibition of Ezh2 enhances commitment of osteoblast progenitors in vitro and bone formation in vivo. Here, we examined the mechanistic effects of Tazemetostat (EPZ6438), an Food and Drug Administration approved Ezh2 inhibitor for epithelioid sarcoma treatment, because this drug could potentially be repurposed to stimulate osteogenesis for clinical indications. We find that Tazemetostat reduces H3K27me3 marks in bivalent domains in enhancers required for bone formation and stimulates maturation of MC3T3 preosteoblasts. Furthermore, Tazemetostat activates bivalent genes associated with the Wingless/integrated (WNT), adenylyl cyclase (cAMP), and Hedgehog (Hh) signaling pathways based on transcriptomic (RNA-seq) and epigenomic (chromatin immunoprecipitation [ChIP]-seq) data. Functional analyses using selective pathway inhibitors and silencing RNAs demonstrate that the WNT and Hh pathways modulate osteogenic differentiation after Ezh2 inhibition. Strikingly, we show that loss of the Hh-responsive transcriptional regulator Gli1, but not Gli2, synergizes with Tazemetostat to accelerate osteoblast differentiation. These studies establish epigenetic cooperativity of Ezh2, Hh-Gli1 signaling, and bivalent regulatory genes in suppressing osteogenesis. Our findings may have important translational ramifications for anabolic applications requiring bone mass accrual and/or reversal of bone loss., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Postnatal cell fate is postulated to be primarily determined by the local tissue microenvironment. Here, we find that Mediator 1 (Med1) dependent epigenetic mechanisms dictate tissue-specific lineage commitment and progression of dental epithelia. Deletion of Med1, a key component of the Mediator complex linking enhancer activities to gene transcription, provokes a tissue extrinsic lineage shift, causing hair generation in incisors. Med1 deficiency gives rise to unusual hair growth via primitive cellular aggregates. Mechanistically, we find that MED1 establishes super-enhancers that control enamel lineage transcription factors in dental stem cells and their progenies. However, Med1 deficiency reshapes the enhancer landscape and causes a switch from the dental transcriptional program towards hair and epidermis on incisors in vivo, and in dental epithelial stem cells in vitro. Med1 loss also provokes an increase in the number and size of enhancers. Interestingly, control dental epithelia already exhibit enhancers for hair and epidermal key transcription factors; these transform into super-enhancers upon Med1 loss suggesting that these epigenetic mechanisms cause the shift towards epidermal and hair lineages. Thus, we propose a role for Med1 in safeguarding lineage specific enhancers, highlight the central role of enhancer accessibility in lineage reprogramming and provide insights into ectodermal regeneration., (© 2023. The Author(s).)

Background: Obesity dysregulates key biological processes underlying the functional homeostasis, fate decisions, and reparative potential of mesenchymal stem/stromal cells (MSCs). Mechanisms directing obesity-induced phenotypic alterations in MSCs remain unclear, but emerging drivers include dynamic modification of epigenetic marks, like 5-hydroxymethylcytosine (5hmC). We hypothesized that obesity and cardiovascular risk factors induce functionally relevant, locus-specific changes in 5hmC of swine adipose-derived MSCs and evaluated their reversibility using an epigenetic modulator, vitamin-C., Methods: Female domestic pigs were fed a 16-week Lean or Obese diet (n = 6 each). MSCs were harvested from subcutaneous adipose tissue, and 5hmC profiles were examined through hydroxymethylated DNA immunoprecipitation sequencing (hMeDIP-seq) followed by an integrative (hMeDIP and mRNA sequencing) gene set enrichment analysis. For clinical context, we compared 5hmC profiles of adipose tissue-derived human MSCs harvested from patients with obesity and healthy controls., Results: hMeDIP-seq revealed 467 hyper- (fold change ≥ 1.4; p-value ≤ 0.05) and 591 hypo- (fold change ≤ 0.7; p-value ≤ 0.05) hydroxymethylated loci in swine Obese- versus Lean-MSCs. Integrative hMeDIP-seq/mRNA-seq analysis identified overlapping dysregulated gene sets and discrete differentially hydroxymethylated loci with functions related to apoptosis, cell proliferation, and senescence. These 5hmC changes were associated with increased senescence in cultured MSCs (p16/CDKN2A immunoreactivity, senescence-associated β-galactosidase [SA-β-Gal] staining), were partly reversed in swine Obese-MSCs treated with vitamin-C, and shared common pathways with 5hmC changes in human Obese-MSCs., Conclusions: Obesity and dyslipidemia are associated with dysregulated DNA hydroxymethylation of apoptosis- and senescence-related genes in swine and human MSCs, potentially affecting cell vitality and regenerative functions. Vitamin-C may mediate reprogramming of this altered epigenomic landscape, providing a potential strategy to improve the success of autologous MSC transplantation in obese patients., (© 2023. The Author(s).)

The title compound, [Mn(C 7 H 7 )(C 6 H 5 O 2 )]PF 6 or [(Cht)Mn(Cp'CO 2 H)]PF 6 , with Cht = cyclo-hepta-trienyl and Cp' = C 5 H 4 , is an air-stable, purple, heteroleptic, cationic sandwich complex with manganese in oxidation state +I and π-coordinating cyclo-hepta-trienyl and cyclo-penta-dienyl ligands. The latter ligand carries the carb-oxy-lic acid functionality. This 'tromancenium-8-carb-oxy-lic acid' with hexa-fluorido-phosphate as counter-ion represents a rare case of a cationic carb-oxy-lic acid. Structurally, this organometallic carb-oxy-lic acid displays the common motif of planar O sp 2 ⋯H-O sp 3 /O sp 3 -H⋯O sp 2 hydrogen-bonded carb-oxy-lic acid dimers with anti -oriented metallocenyl moieties, the cationic charge of which is balanced by octa-hedrally shaped hexa-fluorido-phosphate anions. Positional disorder is observed in the cyclo-hepta-trienyl ring and the PF 6 - anion., (© Thaler et al. 2023.)

Arthrofibrosis, which is characterized by excessive scar tissue and limited motion, can complicate the daily functioning of patients after total knee arthroplasty (TKA). Molecular hallmarks of arthrofibrosis include pathologic accumulation of myofibroblasts and disproportionate collagen deposition. Epigenetic mechanisms, including posttranslation modification of histones, control gene expression and may regulate fibrotic events. This study assessed the role of the bromodomain and extra-terminal (BET) proteins on myofibroblast differentiation. This group of epigenetic regulators recognize acetylated lysines and are targeted by a class of drugs known as BET inhibitors. RNA-seq analysis revealed robust mRNA expression of three BET members (BRD2, BRD3, and BRD4) while the fourth member (BRDT) is not expressed in primary TKA knee outgrowth fibroblasts. RT-qPCR and western blot analyses revealed that BET inhibition with the small molecule JQ1 impairs TGFβ1-induced expression of ACTA2, a key myofibroblast marker, in primary outgrowth knee fibroblasts. Similarly, JQ1 administration also reduced COL3A1 mRNA levels and collagen deposition as monitored by picrosirius red staining. Interestingly, the inhibitory effects of JQ1 on ACTA2 mRNA and protein expression, as well as COL3A1 expression and collagen deposition, were paralleled by siRNA-mediated depletion of BRD4. Together, these data reveal that BRD4-mediated epigenetic events support TGFβ1-mediated myofibroblast differentiation and collagen deposition as seen in arthrofibrosis. To our knowledge, these are the first studies that assess epigenetic regulators and their downstream events in the context of arthrofibrosis. Future studies may reveal clinical utility for drugs that target epigenetic pathways, specifically BET proteins, in the prevention and treatment of arthrofibrosis., (© 2023 Wiley Periodicals LLC.)

Bone formation is controlled by histone modifying enzymes that regulate post-translational modifications on nucleosomal histone proteins and control accessibility of transcription factors to gene promoters required for osteogenesis. Enhancer of Zeste homolog 2 (EZH2/Ezh2), a histone H3 lysine 27 (H3K27) methyl transferase, is a suppressor of osteoblast differentiation. Ezh2 is regulated by SET and MYND domain-containing protein 2 (SMYD2/Smyd2), a lysine methyltransferase that modifies both histone and non-histone proteins. Here, we examined whether Smyd2 modulates Ezh2 suppression of osteoblast differentiation. Musculoskeletal RNA-seq data show that SMYD2/Smyd2 is the most highly expressed SMYD/Smyd member in human bone tissues and mouse osteoblasts. Smyd2 loss of function analysis in mouse MC3T3 osteoblasts using siRNA depletion enhances proliferation and calcium deposition. Loss of Smyd2 protein does not affect alkaline phosphatase activity nor does it result in a unified expression response for standard osteoblast-related mRNA markers (e.g., Bglap, Ibsp, Spp1, Sp7), indicating that Smyd2 does not directly control osteoblast differentiation. Smyd2 protein depletion enhances levels of the osteo-suppressive Ezh2 protein and H3K27 trimethylation (H3K27me3), as expected from increased cell proliferation, while elevating the osteo-inductive Runx2 protein. Combined siRNA depletion of both Smyd2 and Ezh2 protein is more effective in promoting calcium deposition when compared to loss of either protein. Collectively, our results indicate that Smyd2 inhibits proliferation and indirectly the subsequent mineral deposition by osteoblasts. Mechanistically, Smyd2 represents a functional epigenetic regulator that operates in parallel to the suppressive effects of Ezh2 and H3K27 trimethylation on osteoblast differentiation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Atherosclerotic renal artery stenosis (ARAS) is associated with irreversible parenchymal renal disease and regenerative stem cell therapies may improve renal outcomes. Hypoxia preconditioning (HPC) may improve the regenerative functions of adipose tissue-derived mesenchymal stem cells (AMSC) by affecting DNA 5-hydroxymethylcytosine (5hmC) marks in angiogenic genes. Here, we investigated using a porcine ARAS model, whether growth of ARAS AMSCs in hypoxia (Hx) versus normoxia (Nx) would enhance renal tissue repair, and comprehensively analyze how HPC modifies DNA hydroxymethylation compared to untreated ARAS and healthy/normal pigs (n=5 each). ARAS pigs exhibited elevated serum cholesterol, serum creatinine and renal artery stenosis, with a concomitant decrease in renal blood flow (RBF) and increased blood pressure (BP) compared to healthy pigs. Renal artery injection of either autologous Nx or Hx AMSCs improved diastolic BP, reduced kidney tissue fibrosis, and inflammation (CD3+ T-cells) in ARAS pigs. In addition, renal medullary hypoxia significantly lowered with Nx but not Hx AMSC treatment. Mechanistically, levels of epigenetic 5hmC marks (which reflect gene activation) estimated using DNA immunoprecipitation technique were elevated in profibrotic and inflammatory genes in ARAS compared with normal AMSCs. HPC significantly reduced 5hmC levels in cholesterol biosynthesis and oxidative stress response pathways in ARAS AMSCs. Thus, autologous AMSCs improve key renovascular parameters and inflammation in ARAS pigs, with HPC mitigating pathological molecular effects on inflammatory and profibrotic genes which may play a role in augmenting regenerative capacity of AMSCs., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Lotteries have been shown to motivate behaviour change in many settings, but their value as a policy tool is relatively untested. We implemented a pre-registered, citywide experiment to test the effects of three high-pay-off, geographically targeted lotteries designed to motivate adult Philadelphians to get their COVID-19 vaccine. In each drawing, the residents of a randomly selected 'treatment' zip code received half the lottery prizes, boosting their chances of winning to 50×-100× those of other Philadelphians. The first treated zip code, which drew considerable media attention, may have experienced a small bump in vaccinations compared with the control zip codes: average weekly vaccinations rose by an estimated 61 per 100,000 people per week (+11%). After pooling the results from all three zip codes treated during our six-week experiment, however, we do not detect evidence of any overall benefits. Furthermore, our 95% confidence interval provides a 9% upper bound on the net benefits of treatment in our study., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Vitamin C deficiency disrupts the integrity of connective tissues including bone. For decades this function has been primarily attributed to Vitamin C as a cofactor for collagen maturation. Here, we demonstrate that Vitamin C epigenetically orchestrates osteogenic differentiation and function by modulating chromatin accessibility and priming transcriptional activity. Vitamin C regulates histone demethylation (H3K9me3 and H3K27me3) and promotes TET-mediated 5hmC DNA hydroxymethylation at promoters, enhancers and super-enhancers near bone-specific genes. This epigenetic circuit licenses osteoblastogenesis by permitting the expression of all major pro-osteogenic genes. Osteogenic cell differentiation is strictly and continuously dependent on Vitamin C, whereas Vitamin C is dispensable for adipogenesis. Importantly, deletion of 5hmC-writers, Tet1 and Tet2, in Vitamin C-sufficient murine bone causes severe skeletal defects which mimic bone phenotypes of Vitamin C-insufficient Gulo knockout mice, a model of Vitamin C deficiency and scurvy. Thus, Vitamin C's epigenetic functions are central to osteoblastogenesis and bone formation and may be leveraged to prevent common bone-degenerating conditions., (© 2022. The Author(s).)

High-throughput microRNA sequencing was performed during differentiation of MC3T3-E1 osteoblasts to develop working hypotheses for specific microRNAs that control osteogenesis. The expression data show that miR-101a, which targets the mRNAs for the epigenetic enzyme Ezh2 and many other proteins, is highly upregulated during osteoblast differentiation and robustly expressed in mouse calvaria. Transient elevation of miR-101a suppresses Ezh2 levels, reduces tri-methylation of lysine 27 in histone 3 (H3K27me3; a heterochromatic mark catalyzed by Ezh2), and accelerates mineralization of MC3T3-E1 osteoblasts. We also examined skeletal phenotypes of an inducible miR-101a transgene under direct control of doxycycline administration. Experimental controls and mir-101a over-expressing mice were exposed to doxycycline in utero and postnatally (up to 8 weeks of age) to maximize penetrance of skeletal phenotypes. Male mice that over-express miR-101a have increased total body weight and longer femora. MicroCT analysis indicate that these mice have increased trabecular bone volume fraction, trabecular number and trabecular thickness with reduced trabecular spacing as compared to controls. Histomorphometric analysis demonstrates a significant reduction in osteoid volume to bone volume and osteoid surface to bone surface. Remarkably, while female mice also exhibit a significant increase in bone length, no significant changes were noted by microCT (trabecular bone parameters) and histomorphometry (osteoid parameters). Hence, miR-101a upregulation during osteoblast maturation and the concomitant reduction in Ezh2 mediated H3K27me3 levels may contribute to the enhanced trabecular bone parameters in male mice. However, the sex-specific effect of miR-101a indicates that more intricate epigenetic mechanisms mediate physiological control of bone formation and homeostasis., (© 2022. The Author(s).)

In continuation of our study of the chemistry of cationic (cycloheptatrienyl)(cyclopentadienyl)manganese(I) sandwich complexes, so-called "tromancenium" salts, we report here on their boron-substituted derivatives focusing on useful boron-mediated synthetic applications. Transmetalation of lithiated tricarbonyl(cyclopentadienyl)manganese ("cymantrene") with boric or diboronic esters affords monoborylated cymantrenes that are converted by advanced high-power LED photosynthesis followed by oxidation with tritylium to their 8-boron-substituted tromancenium complexes. These new functionalized tromancenium salts are fully characterized by 1 H/ 11 B/ 13 C/ 19 F/ 55 Mn NMR, IR, UV-vis, HRMS spectroscopy, single-crystal structure analysis (XRD) and cyclic voltammetry (CV). IR spectra were thoroughly analyzed by density functional theory (DFT) on the harmonic approximation in qualitative agreement of calculated vibrations with experimental values. Uncommon chemical reactivity of these borylated tromancenium salts is observed, due to the strongly electron-withdrawing cationic tromancenium moiety. No Suzuki-type cross-coupling reactions proved so far achievable, but unusual copper-promoted amination with sodium azide under microwave irradiation is possible. Diazoniation of aminotromancenium affords an extremely reactive dicationic tromanceniumdiazonium salt, which is too labile for standard Sandmeyer reactions, in contrast to analogous chemistry of cobaltocenium salts. Overall, borylated tromancenium salts display unexpected and intriguing chemical properties with the potential for novel synthetic applications in future work., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

One pivotal topic on habitat selection is to understand the influence of intrinsic and extrinsic factors on the selection process. Although the effect of some extrinsic variables and sex has been extensively studied, almost nothing is known about the effect of personality, particularly on snakes. Here we addressed the relative importance of extrinsic (tree-related) and intrinsic (sex and personality) factors driving microhabitat selection decisions of a nocturnal tree snake (Leptodeira annulata). We implemented a protocol to quantify the influence of personality and sex on the role of extrinsic variables on microhabitat selection. First, we conducted three behavioral experiments to extract the shyness-boldness and avoidance-exploitation personality traits of male snakes. Then, we evaluated the role of sex and personality on the effect of tree-traits (thickness, canopy cover and shelter availability) on microhabitat selection, using two-step conditional logistic regression. Snakes consistently selected tree trunks, preferably thick and with high canopy cover and shelter availability, independently of their sex or personality. For this species, only extrinsic variables determined microhabitat decisions. Our protocol will aid to quantify the role of personality on microhabitat selection of other species and understand whether this is an important variable in habitat decision-making or not., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Scattered tubular-like cells (STCs) are dedifferentiated renal tubular cells endowed with progenitor-like characteristics to repair injured parenchymal cells. STCs may be damaged and rendered ineffective by renal artery stenosis (RAS), but the underlying processes remain unclear. We hypothesized that RAS alters the epigenetic landscape on DNA and the ensuing gene transcriptional profile of swine STCs., Methods: CD24+/CD133+ STCs were isolated from pig kidneys after 10 weeks of RAS or sham ( n = 3 each) and their whole 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) profiles were examined by 5mC and 5hmC immunoprecipitation sequencing (MeDIP-/hMeDIP-seq, respectively). A subsequent integrated (MeDIP/hMeDIP-seq/mRNA-seq) analysis was performed by comparing all online available gene sets using Gene Set Enrichment Analysis. Apoptosis, proteolysis, and mitochondrial structure and function were subsequently evaluated in vitro., Results: Differential expression (DE) analysis revealed 239 genes with higher and 236 with lower 5mC levels and 275 genes with higher and 315 with lower 5hmC levels in RAS-STCs compared to Normal-STCs (fold change ≥1.4 or ≤0.7, p ≤ 0.05). Integrated MeDIP-/hMeDIP-seq/mRNA-seq analysis identified several overlapping (DE-5mC/mRNA and DE-5hmC/mRNA levels) genes primarily implicated in apoptosis, proteolysis, and mitochondrial functions. Furthermore, RAS-STCs exhibited decreased apoptosis, mitochondrial matrix density, and ATP production, and increased intracellular amino acid concentration and ubiquitin expression., Conclusions: Renal ischemia induces epigenetic changes in apoptosis-, proteolysis-, and mitochondria-related genes, which correlate with alterations in the transcriptomic profile and corresponding function of swine STCs. These observations may contribute to developing novel targeted interventions to preserve the reparative potency of STCs in renal disease.

Arthrofibrosis is characterized by excessive extracellular matrix (ECM) deposition that results in restricted joint motion after total knee arthroplasties (TKAs). Currently, treatment options are limited. Therefore, an in vitro model of knee-related myofibroblastogenesis is valuable to facilitate investigation of the arthrofibrotic process, diagnostic and therapeutic options. In this study, we obtained intraoperative posterior capsule (PC), quadriceps tendon (QT), and suprapatellar pouch (SP) tissues from the knees of four patients undergoing primary TKAs for osteoarthritis. From these tissues, we isolated primary cells by the outgrowth method and subsequently characterized these cells in the absence and presence of the pro-myofibroblastic cytokine, transforming growth factor beta 1 (TGFβ1). Light microscopy of knee outgrowth cells revealed spindle-shaped cells, and immunofluorescence (IF) analysis demonstrated staining for the fibroblast-specific markers TE-7 and vimentin (VIM). These knee outgrowth fibroblasts differentiated readily into myofibroblasts as reflected by enhanced α-smooth muscle actin (ACTA2) mRNA and protein expression and increased mRNA expression of collagen type 1 (COL1A1) and type 3 (COL3A1) with collagenous matrix deposition in the presence of TGFβ1. Outgrowth knee fibroblasts were more sensitive to TGFβ1-mediated myofibroblastogenesis than adipose-derived mesenchymal stromal/stem cells (MSCs). While outgrowth knee fibroblasts isolated from three anatomical regions in four patients exhibited similar gene expression, these cells are distinct from other fibroblastic cell types (i.e., Dupuytren's fibroblasts) as revealed by RNA-sequencing. In conclusion, our study provides an in vitro myofibroblastic model of outgrowth knee fibroblasts derived from patients undergoing primary TKA that can be utilized to study myofibroblastogenesis and assess therapeutic strategies for arthrofibrosis., (© 2022 Wiley Periodicals LLC.)

Aims: Outcomes of current operative treatments for arthrofibrosis after total knee arthroplasty (TKA) are not consistently positive or predictable. Pharmacological in vivo studies have focused mostly on prevention of arthrofibrosis. This study used a rabbit model to evaluate intra-articular (IA) effects of celecoxib in treating contracted knees alone, or in combination with capsular release., Methods: A total of 24 rabbits underwent contracture-forming surgery with knee immobilization followed by remobilization surgery at eight weeks. At remobilization, one cohort underwent capsular release (n = 12), while the other cohort did not (n = 12). Both groups were divided into two subcohorts (n = 6 each) - one receiving IA injections of celecoxib, and the other receiving injections of vehicle solution (injections every day for two weeks after remobilization). Passive extension angle (PEA) was assessed in live rabbits at 10, 16, and 24 weeks, and disarticulated limbs were analyzed for capsular stiffness at 24 weeks., Results: IA celecoxib resulted in greater mean PEA at ten weeks (69.6° (SD 4.6) vs 45.2° (SD 9.6), p = 0.004), 16 weeks (109.8° (SD 24.2) vs 60.9° (SD10.9), p = 0.004), and 24 weeks (101.0° (SD 8.0) vs 66.3° (SD 5.8), p = 0.004). Capsular stiffness was significantly reduced with IA celecoxib (2.72 Newton per cm (N·cm)/° (SD 1.04), p = 0.008), capsular release (2.41 N·cm/° (SD 0.80), p = 0.008), and capsular release combined with IA celecoxib (3.56 N·cm/° (SD 0.99), p = 0.018) relative to IA vehicle (6.09 N·cm/° (SD 1.64))., Conclusion: IA injections of a celecoxib led to significant improvements in passive extension angles, with reduced capsular stiffness, when administered to rabbit knees with established experimental contracture. Celecoxib was superior to surgical release, and the combination of celecoxib and a surgical release did not provide any additional value. Cite this article: Bone Joint Res  2022;11(1):32-39.

Differentiation of multi-potent mesenchymal stromal cells (MSCs) is directed by the activities of lineage-specific transcription factors and co-factors. A subset of these proteins controls the accessibility of chromatin by recruiting histone acetyl transferases or deacetylases that regulate acetylation of the N-termini of H3 and H4 histone proteins. Bromodomain (BRD) proteins recognize these acetylation marks and recruit the RNA pol II containing transcriptional machinery. Our previous studies have shown that Brd4 is required for osteoblast differentiation in vitro. Here, we investigated the role of Brd4 on endochondral ossification in C57BL/6 mice and chondrogenic differentiation in cell culture models. Conditional loss of Brd4 in the mesenchyme (Brd4 cKO, Brd4 fl/fl : Prrx1-Cre) yields smaller mice that exhibit alteration in endochondral ossification. Importantly, abnormal growth plate morphology and delayed long bone formation is observed in juvenile Brd4 cKO mice. One week old Brd4 cKO mice have reduced proliferative and hypertrophic zones within the physis and exhibit a delay in the formation of the secondary ossification center. At the cellular level, Brd4 function is required for chondrogenic differentiation and maturation of both ATDC5 cells and immature mouse articular chondrocytes. Mechanistically, Brd4 loss suppresses Sox9 levels and reduces expression of Sox9 and Runx2 responsive endochondral genes (e.g., Col2a1, Acan, Mmp13 and Sp7/Osx). Collectively, our results indicate that Brd4 is a key epigenetic regulator required for normal chondrogenesis and endochondral ossification., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Metal orthopedic implants are largely biocompatible and generally achieve long-term structural fixation. However, some orthopedic implants may loosen over time even in the absence of infection. In vivo fixation failure is multifactorial, but the fundamental biological defect is cellular dysfunction at the host-implant interface. Strategies to reduce the risk of short- and long-term loosening include surface modifications, implant metal alloy type, and adjuvant substances such as polymethylmethacrylate cement. Surface modifications (e.g., increased surface rugosity) can increase osseointegration and biological ingrowth of orthopedic implants. However, the localized responses of cells to implant surface modifications need to be better characterized. As an in vitro model for investigating cellular responses to metallic orthopedic implants, we cultured mesenchymal stromal/stem cells on clinical-grade titanium disks (Ti6Al4V) that differed in surface roughness as high (porous structured), medium (grit blasted), and low (bead blasted). Topological characterization of clinically relevant titanium (Ti) materials combined with differential mRNA expression analyses (RNA-seq and real-time quantitative polymerase chain reaction) revealed alterations to the biological phenotype of cells cultured on titanium structures that favor early extracellular matrix production and observable responses to oxidative stress and heavy metal stress. These results provide a descriptive model for the interpretation of cellular responses at the interface between native host tissues and three-dimensionally printed modular orthopedic implants, and will guide future studies aimed at increasing the long-term retention of such materials after total joint arthroplasty. Impact statement Using an in vitro model of implant-to-cell interactions by culturing mesenchymal stromal cells (MSCs) on clinically relevant titanium materials of varying topological roughness, we identified mRNA expression patterns consistent with early extracellular matrix (ECM) production and responses to oxidative/heavy metal stress. Implants with high surface roughness may delay the differentiation and ECM formation of MSCs and alter the expression of genes sensitive to reactive oxygen species and protein kinases. In combination with ongoing animal studies, these results will guide future studies aimed at increasing the long-term retention of widely used titanium materials after total joint arthroplasty.

Skeletal development and bone formation are regulated by epigenetic mechanisms that either repress or enhance osteogenic commitment of mesenchymal stromal/stem cells and osteoblasts. The transcriptional suppressive trimethylation of histone 3 lysine 27 (H3K27me3) hinders differentiation of pre-committed osteoblasts. Osteoblast maturation can be stimulated by genetic loss of the H3K27 methyltransferase Ezh2 which can also be mimicked pharmacologically using the classical Ezh2 inhibitor GSK126. Identification of other Ezh2 inhibitors (iEzh2) that enhance osteogenic potential would increase chemical options for developing new bone stimulatory compounds. In this study, we examined a panel of iEzh2s and show that all eight inhibitors we tested are capable of accelerating osteoblast differentiation to different degrees at concentrations that are well below cytotoxic concentrations. Inhibition of Ezh2 is commensurate with loss of cellular H3K27me3 levels while forced expression of Ezh2 reverses the effect of Ezh2 suppression. Reduced Ezh2 function by siRNA depletion of Ezh2 mRNA and protein levels also stimulates osteoblastogenesis, consistent with the specificity of iEzh2 to target the active site of Ezh2. Diminished Ezh2 levels preempt the effects of iEzh2s on H3K27me3. GSK126, EPZ-6438 and siRNA depletion of Ezh2 each are effective in reducing H3K27me3 levels. However, EPZ-6438 is more potent than GSK126 in stimulating osteoblastogenesis, as reflected by increased extracellular matrix mineralization. Collectively, our data indicate that Ezh2 inhibitors properly target Ezh2 consistent with their biochemical affinities. The range of compounds capable of promoting osteogenesis presented in this study offers the opportunity to develop diverse bone anabolic strategies for distinct clinical scenarios, including spine fusion, non-union of bone and dental implant enhancement., (Copyright © 2021 Elsevier Inc. All rights reserved.)